Dual-AAV Vector-Based Systems and Methods for Delivering Oversized Genes to Mammalian Cells

ABSTRACT

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of PCT Intl. Patent Appl. No.PCT/US2012/065645 filed Nov. 16, 2012 (nationalized; Atty. Dkt. No.36689.353), which claimed priority to U.S. Provisional Patent Appl. No.61/560,437, filed Nov. 16, 2011 (expired); the contents of each of whichis hereby incorporated in its entirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersEY00331 and EY021721 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology and virology, and in particular, to the development of genedelivery vehicles. Disclosed are improved rAAV dual-vector systems, andcompositions useful in delivering a variety of nucleic acid segments,including those encoding therapeutic proteins polypeptides, peptides,antisense oligonucleotides, or ribozyme constructs to selected hostcells for use in various gene-therapy regimens. Methods are alsoprovided for preparing and using these modified rAAV-dual-vector basedsystems in a variety of viral-based gene therapies, and in particular,for the treatment and/or amelioration of symptoms of MyosinVII-deficiency, including, without limitation, the treatment of humanUsher syndrome.

2. Description of Related Art

As has been established by a multitude of successful proof-of-conceptstudies, and various clinical trials, recombinant AAV has emerged as themost optimal gene delivery vehicle to treat retinal disease. However,one limitation of AAV is its relatively small DNA packagingcapacity—approximately 4.7 kilobases (KB). Thus, standard AAV vectorsystems are unsuitable for addressing diseases in which large genes aremutated or otherwise dysfunctional. An example of such a disease isUsher syndrome.

The most common form of Usher syndrome, USH1B, is a severeautosomal-recessive, deaf-blindness disorder caused by mutations in themyosinVIIa gene. Blindness occurs from a progressive retinaldegeneration that begins after deafness, and after development of theretina. MYO7a protein is expressed in photoreceptors and retinal pigmentepithelium (RPE), and is involved in opsin transport throughphotoreceptor cilia and the movement of RPE melanosomes.

The coding region for the human Myosin VII protein (MYO7a), however, is6534 or 6648 nucleotides in length (depending on the allelic variant),making traditional AAV vector systems unsuitable for gene therapy ofUSH1B.

Previously, Allocca et al. (2008) published intriguing resultssuggesting that AAV5 serotype vectors were capable of packaging genomesof up to 8.9 KB in size, and that these vectors expressed full-lengthproteins when delivered in vivo. In this study, the authors expressedfull-length MYO7A protein from an AAV5 vector containing the CMVpromoter driving hMyo7a. Subsequent studies, carried out to directlyvalidate the Allocca et al. findings were simultaneously published bythree independent groups (Lai et al., 2010; Dong et al., 2010; Wu etal., 2010), and their publication was accompanied by an expertcommentary (See, Hirsch et al., 2010). While all three studies confirmedthat these ‘oversized’ AAV5 vectors did indeed drive full-length proteinexpression, the genetic content of each vector capsid was found to belimited only to ˜5 KB of DNA, and not the 8.7 KB originally reported byAllocca et al. (2008). These vector capsids were shown to contain a“heterogeneous mixture” of truncated vector genomes (e.g., the 5′-end ofthe gene, the 3′-end of the gene, or a mixture of the two, with aninternal sequence deletion) (Lai et al., 2010; Dong et al., 2010; Wu etal., 2010). Additionally, these oversized/heterogeneous vectorsexhibited poor packaging efficiency (i.e., low-vector titers) and lowtransduction efficiency when compared to matched reporter vectors ofstandard size (<5 KB) (Wu et al., 2010).

Using this ‘heterogeneous’ system, vectors containing portions of theMYO7A transgene were packaged, however, and proof-of-concept resultswere demonstrated in the shaker-1 mouse model of USH1B. The therapeuticresults achieved with the heterogeneous AAV-hMyo7a vectors werecomparable to previous gene replacement results using a Lentivirus-basedhMyo7a vector (Hashimoto et al., 2007).

This Lentivirus-Myo7a vector is under development by Oxford BioMedica incollaboration with Sanofi-Aventis for a phase I/II clinical trial ofUSH1B, marketed under the name UshStat® LentiVector®. Lentivirus isregarded as a vector platform that is not well-suited for infectingpost-mitotic (i.e., non-dividing) cells. Furthermore, although thevector is suitable for transducing RPE, many studies have shown it to beineffective at transducing adult photoreceptors. Even though MYO7A isexpressed in both cell types, UshStat® may only be effective at rescuingthe RPE phenotype. A study showed that PRs are actually the initial siteof disease, so not targeting this cell type effectively may result inzero therapy, although it remains to be seen in the human clinicaltrial.

Because of the excellent safety profile and encouraging reports ofefficacy in the AAV gene therapy trials for LCA2/RPE65, there has beencontinuing interest in creating an AAV-based system for treating USH1Bpatients. However, the current AAV vector for MYO7A, as previouslymentioned, is heterogeneous; it is manufactured and purified as asingle-virus preparation containing a mixture of viral payloads. Thisfact, unfortunately, makes it virtually impossible to characterize thevector fully—a requirement for government regulatory review andapproval. In order to address this concern directly (i.e., that thevector genome of any AAV-based vectors for USH1B must be fullycharacterized before gaining Food and Drug Administration (FDA)approval, the inventors have developed an AAV dual-vector-based systemto facilitate gene therapy approaches for treating USH1B.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for gene therapy ofdiseases, such as Usher syndrome 1B (USH1B). The inventors havecharacterized dual vector platforms described herein as to transcriptfidelity, and have shown and that mRNA arising from the system is 100%accurate relative to what would be predicted by correct homologousrecombination on the front and back vector pairs, making them useful asgene therapy delivery vector systems.

One aspect of the invention concerns a dual AAV vector system thatpermits expression of full-length proteins, whose coding sequenceexceeds the polynucleotide packaging capacity of an individual AAVvector.

In one embodiment, a vector system of the invention comprises:

(i) a first AAV vector polynucleotide comprising an inverted terminalrepeat at each end of the polynucleotide, and a suitable promoterfollowed by a partial coding sequence that encodes an N-terminal part ofa selected full-length polypeptide; and

(ii) a second AAV vector polynucleotide comprising an inverted terminalrepeat at each end of the polynucleotide, and a partial coding sequencethat encodes a C-terminal part of the selected full-length polypeptide,optionally followed by a polyadenylation (pA) signal sequence.

The coding sequence in the first and second AAV vectors comprisessequence that overlaps, such that part of the coding sequence present atthe 3′-end of the coding sequence of the first vector is identical orsubstantially identical with part of the coding sequence present at the5′-end of the coding sequence of the second vector. In an illustrativeembodiment, the polypeptide encoded is a wild type orbiologically-functional human myosin VIIa polypeptide (hmyo7a).

In another embodiment, a vector system of the invention includes:

-   -   (i) a first AAV vector polynucleotide comprising an inverted        terminal repeat at each end, a suitable promoter followed by a        partial coding sequence that encodes an N-terminal part of a        selected full-length polypeptide followed by a splice donor site        for an intron and an intron; and    -   (ii) a second AAV vector polynucleotide comprising an inverted        terminal repeat at each end, followed by an intron and a splice        acceptor site for the intron, followed by a partial coding        sequence that encodes a C-terminal part of the selected        full-length polypeptide, optionally followed by a        polyadenylation (pA) signal sequence.

The intron sequence in the first and second AAV vectors includes asequence that overlaps, such that all or part of the intron sequencepresent at the 3′-end of the coding sequence of the first vector isidentical, or substantially identical, with all or part of the intronsequence present at the 5′-end of the coding sequence of the secondvector. In one embodiment, the intron is intron 23 of the hmyo7a gene.In a specific embodiment, the polypeptide encoded is a wild type (i.e.,functional) human myosin VIIa polypeptide, and the intron is the fullintron 23 of the hmyo7a gene.

BRIEF DESCRIPTION OF THE DRAWINGS

For promoting an understanding of the principles of the invention,reference will now be made to the embodiments, or examples, illustratedin the drawings and specific language will be used to describe the same.It will, nevertheless be understood that no limitation of the scope ofthe invention is thereby intended. Any alterations and furthermodifications in the described embodiments, and any further applicationsof the principles of the invention as described herein are contemplatedas would normally occur to one of ordinary skill in the art to which theinvention relates.

The following drawings form part of the present specification and areincluded to demonstrate certain aspects of the present invention. Theapplication contains at least one drawing that is executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Patent and Trademark Office uponrequest and payment of the necessary fee. The invention may be betterunderstood by reference to the following description taken inconjunction with the accompanying drawings, in which like referencenumerals identify like elements, and in which:

FIG. 1 shows the formation of complete gene cassette from dual AAVvectors via homologous recombination;

FIG. 2 shows a schematic of the two vector components that make upDual-Vector System 1 in accordance with one aspect of the presentinvention;

FIG. 3 shows a schematic of the two vector components that make upDual-Vector System 2 in accordance with one aspect of the presentinvention. Native hMyo7a intron 23 in shown in light green; splice donorand splice acceptor sequence are shown in dark green;

FIG. 4 shows the schematic of the two vector components that make upDual-Vector System 3 in accordance with one aspect of the presentinvention. This is an exemplary standard transplicing dual vector pairswith the “intron” in this case referring to the alkaline phosphatasesplice donor and acceptor sites. Synthetic alkaline phosphatase (AP)intron is shown in light blue; AP splice donor and splice acceptorsequences are shown in dark blue;

FIG. 5 shows an immunoblot to detect the presence of MYO7A in infectedor transfected HEK293 cells. Heterogeneous vectors (described inintroduction) are compared to all three dual-vector systems. Dualvectors were packaged either in AAV2 or in AAV2(triple mutant) capsids.The triple mutant contains three tyrosine-to-phenylalanine mutations onthe capsid surface. For all three dual-vector systems, infections wereperformed with either a) the front-half (N-terminal) and back-half(C-terminal) vectors; or b) the front-half vectors alone (to confirm thepresence or absence of a truncated protein product expressed from thepromoter-containing N-terminal vectors);

FIG. 6A and FIG. 6B show immunoblot to detect the presence of myo7A inHEK293 cells infected with Dual-Vector System 1. Results are presentedas a time course from 3-7 days post infection (lanes 3-7) and arecompared to cells transfected with myo7a plasmid (lane 1) and uninfectedcontrol (lane 2). The area inside the white box shown in FIG. 6A ismagnified and presented at higher contrast in FIG. 6B. Starting at3-days' post-infection, full-length human MYO7a protein was visible,with peak expression occurring around day 5;

FIG. 7A and FIG. 7B show retinas from untreated mice and mice treatedsubretinally with Dual-Vector System 1. Immunohistochemistry (IHC) wasperformed using an antibody directed against MYO7A. The ‘green’ is thestain of MYO7A and ‘blue’ corresponds to nuclear, DAPI stain;

FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show differences in RPEmelanosome localization in wild type vs. shaker-1 mice. In wild typemice, RPE melanosome apically migrate towards photoreceptor outersegments (FIG. 8A) whereas this phenomenon fails to occur in micelacking Myo7a (shaker-1), as seen in (FIG. 8B). To the right is a highmagnification image of single RPE cells from either a wild type (FIG.8C) or shaker-1 (FIG. 8D) mouse showing this phenomenon up close;

FIG. 9A, FIG. 9B, and FIG. 9C show apical migration of RPE melanosomesis restored in shaker-1 mice injected with Dual-Vector System 1.Electron microscopy reveals that melanosomes of untreated shaker-1 micedo not apically migrate (FIG. 9A). In shaker-1 mice injected withDual-Vector System 1 (packaged in AAV2), RPE melanosomes migrateapically towards photoreceptors, which can be seen here in both low- andhigh-magnification images (FIG. 9B and FIG. 9C);

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, and FIG. 10Fillustrate the expression of MYO7A from single AAV2 and AAV5 vectors incultured cells. FIG. 10A is a diagram of the viral vector encoding humanMYO7A cDNA. FIG. 10B is a western blot of WT eyecup (lane 1), primaryRPE cultures derived from Myo7a-null mice and infected with AAV2-MYO7A(lane 2) or AAV5-MYO7A (lane 3), or not infected (lane 4), and primaryRPE cultures derived from Myo7a^(+/−) mice (lane 5). All lanes wereimmunolabeled with antibodies against actin (as a loading indicator ofrelative protein loading) and MYO7A. FIG. 10C, FIG. 10D, FIG. 10E, andFIG. 10F are immunofluorescence images of primary RPE cell cultures.Cells derived from Myo7a-null mice that were not infected (FIG. 10C),from Myo7a^(+/−) mice (FIG. 10D), or from Myo7a-null mice infected witheither 1×AAV2-MYO7A (FIG. 10E) or 1×AAV5-MYO7A (FIG. 10F). Scale=10 μm;

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11F-1,FIG. 11G, FIG. 11G-1, FIG. 11H, FIG. 11I, FIG. 11J, FIG. 11K, FIG. 11L,and FIG. 11M show the expression of MYO7A from single AAV2 and AAV5vectors in vivo. FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11Eshow EM images of MYO7A immunogold labelling of the connecting ciliumand pericilium from rod photoreceptors in a Myo7a-null retina. FIG. 11Ais a longitudinal section from an untreated Myo7a-null retina(background label only). FIG. 11B and FIG. 11C are longitudinal sectionsfrom Myo7a-null retinas treated with AAV2-MYO7A (FIG. 11B) or AAV5-MYO7A(FIG. 11C). Scale=50 nm. FIG. 11D and FIG. 11E are transverse sectionsof connecting cilia from rod photoreceptors in Myo7a-null retinastreated with AAV2-MYO7A (FIG. 11D) or AAV5-MYO7A (FIG. 11E). Scale=50nm. FIG. 11F and FIG. 11G show EM images of RPE cells from Myo7a-nullretinas treated with AAV2-MYO7A (FIG. 11F) or AAV5-MYO7A (FIG. 11G).Scale=500 nm. BM=Bruch's membrane, AP=apical processes. Areas indicatedby rectangles are enlarged in FIG. 11F-1 and FIG. 11G-1 to show MYO7Aimmunogold labeling (indicated by circles). Scale=50 nm. FIG. 11H andFIG. 11I show EM image of a longitudinal section of the connectingcilium and pericilium from a rod (FIG. 11H) and a cone (FIG. 11I)photoreceptor in a Myo7a-null retina, treated with AAV2-MYO7A. Thesection was double-labeled with MYO7A (12-nm gold) and rod opsin (15-nmgold) antibodies. Rod outer segments were labeled with the opsinantibody, while cones were identified by lack of rod opsin labeling intheir outer segments. The sections show just the base of the outersegments. Nearly all the label in the connecting cilium is MYO7A, evenin the rod. Scale=50 nm. FIG. 11J, FIG. 11K, FIG. 11L, and FIG. 11M arebar graphs indicating MYO7A immunogold particle density in the rodphotoreceptor cilium and pericilium (FIG. 11J and FIG. 11K) and in theRPE (FIG. 11L and FIG. 11M), following treatment with AAV2-MYO7A (FIG.11J and FIG. 11L) or AAV5-MYO7A (FIG. 11K and FIG. 11M) of differentconcentrations. n=3 animals per condition. Bars indicate SEM;

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, and FIG. 12F showcorrection of melanosome localization, following subretinal injectionswith AAV2-MYO7A or AAV5-MYO7A. Light micrographs showing the presence ofmelanosomes in the apical processes of the RPE in a WT retina (FIG. 12A)and retinas injected with AAV2-MYO7A (FIG. 12B) or AAV5-MYO7A (FIG.12C). Further away from the injection site (FIG. 12D), melanosomes arepresent in the apical processes of some RPE cells, but not in others(arrows indicate apical melanosomes; white lines indicate regions wheremelanosomes are absent from the apical processes). FIG. 12E illustratesa region distant from injection site, where all RPE cells lackedmelanosomes in their apical processes. Brackets on left side indicateRPE apical processes. Scale=25 μm. FIG. 12F is a diagram of an eyecup,indicating the relative locations of the images shown in FIG. 12A, FIG.12B, FIG. 12C, FIG. 12D, and FIG. 12E. Arrow indicates the site ofinjection; ONH indicates the optic nerve head;

FIG. 13 shows the correction of abnormal levels of opsin in theconnecting cilium and pericilium of rod photoreceptors, followingsubretinal injections with AAV2-MYO7A or AAV5-MYO7A. The bar graph showsopsin immunogold gold particle density along the length of theconnecting cilium. Ultrathin sections of retinas from Myo7a-null and WTmice were stained with rod opsin antibody. The Myo7a-null retinas hadbeen untreated, or treated with either 1× or 1:100 AAV2-MYO7A orAAV5-MYO7A. n=3 animals per condition. Bars indicate SEM;

FIG. 14A-1, FIG. 14A-2, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG.14F, and FIG. 14G show the expression of MYO7A from the overlappingAAV2-MYO7A dual vectors. FIG. 14A-1 and FIG. 14A-2 illustrate a diagramof the overlapping AAV2-MYO7A dual vectors. The overlapping regioncontains 1365 bases. FIG. 14B is a Western blot of proteins from primaryRPE cultures derived from Myo7a-null mice and not infected (lane 1), orinfected with AAV2-MYO7A(dual) (lane 2); primary RPE cultures derivedfrom Myo7a^(+/−) mice (lane 3); WT eyecup (lane 4); HEK293A cellstransfected with pTR-smCBA-MYO7A (lane 5). All lanes were immunolabeledwith anti-MYO7A and anti-actin. FIG. 14C, FIG. 14D, FIG. 14E, and FIG.14F show immunofluorescence of cultured RPE cells transduced withAAV2-MYO7A(dual). FIG. 14C, FIG. 14D, and FIG. 14E show primary RPEcultures derived from Myo7a-null mice and ARPE19 (FIG. 14F) cells.Scale=10 μm. FIG. 14G is a bar graph indicating the distribution ofMYO7A immunogold particle density among RPE cells from retinas ofMyo7a-null mice, injected with AAV2-MYO7A(dual). n=3 animals;

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F, and FIG. 15Gillustrate correction of mutant phenotypes, following subretinalinjections with AAV2-MYO7A(dual). FIG. 15A is the results of lightmicroscopy of a semi-thin section from a treated Myo7a-null mouseretina. The region shown is near the injection site. Arrows indicatemelanosomes in the apical processes. White lines indicate cells thatstill show the Myo7a-null phenotype, with an absence of melanosomes inthe apical processes. Scale=50 μm. FIG. 15B is a low-magnificationimmunoEM image of RPE from a retina treated with AAV2-MYO7A(dual). As inFIG. 15A, the white line indicated a region that still showed theMyo7a-null phenotype. Rectangle ‘c’, includes melanosomes in the apicalregion, indicating a corrected RPE cell. Scale=500 nm. FIG. 15C, FIG.15D, and FIG. 15E show higher-magnification images of regions outlinedby the rectangles shown in FIG. 15B. MYO7A immunogold particles wereindicated by circles. Scale=50 nm. FIG. 15F is a bar graph illustratingMYO7A immunogold particle density measured in RPE cells from Myo7a-nullretinas, WT retinas, or from Myo7a-null retinas treated withAAV2-MYO7A(dual) and determined to be corrected or not corrected by thelocation of their apical melanosomes. n=3 animals per condition. Barsindicate SEM. FIG. 15G is an immunoEM image of a rod photoreceptorcilium double-labeled with antibodies against MYO7A (small goldparticles) and against rod opsin (large gold particles). MYO7A labelingis associated with the connecting cilium and periciliary membrane,indicating expression and correct localization of MYO7A. While thisregion is devoid of opsin labeling, which is restricted to the diskmembranes, it is consistent with the wild type (WT) phenotype, thusindicating correction of the mutant phenotype. Scale=300 nm;

FIG. 16 shows the validation of dual AAV vectors for delivery offull-length MYO7A in vivo. Immunoblot showing expression of MYO7A(green) in retinas of wild type (C57BL/6) mice (lane 1), heterozygousshaker-1^(+/) mice (lane 2) and shaker-1^(−/−) mice injected with‘simple overlap’ Myo7a vectors packaged in AAV8(733) vectors. BothN-terminal and C-terminal vectors of the ‘simple overlap’ system wereinjected at a concentration of 3×10¹⁰ vector genomes/μL. Dual AAVvectors mediated expression of a MYO7A that was identical in size tothat found in WT and shaker-1^(+/−) mice. β-actin (visualized here inred) was used as a loading control to validate that equal amounts ofprotein were loaded in each well;

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F showAAV-mediated MYO7A expression in ARPE-19. Cells were transduced with1×AAV2-MYO7A (FIG. 17A), AAV5-MYO7A (FIG. 17B), 1/100 dilutions thereof(FIG. 17C and FIG. 17D) and AAV2-MYO7A (dual) (FIG. 17F). Non-transducedcells were used as a control (FIG. 17E); Red, MYO7A; Blue, DAPI.Scale=10 μm;

FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show MYO7A expression in theconnecting cilium and pericilium of rod photoreceptors from Myo7a-nullretinas injected with diluted AAV2-MYO7A (FIG. 18 a and FIG. 18B) orAAV5-MYO7A (FIG. 18C and FIG. 18D); (FIG. 18A and FIG. 18C) 1:10, (FIG.18B and FIG. 18D) 1:100. Scale=200 nm;

FIG. 19 shows the structural preservation of injected Myo7a-nullretinas. Light microscopy of the photoreceptor layer 3 weeks afterinjection with 10×AAV5-MYO7A. Scale=15 μm;

FIG. 20 shows structural preservation of injected Myo7a-null retinas.Light microscopy of photoreceptor layer 3 months after injection with1×AAV2-MYO7A. Scale=10 μm;

FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D show correction of abnormallevels of opsin in the connecting cilium and pericilium of rodphotoreceptors following subretinal injections with AAV2-MYO7A orAAV5-MYO7A. ImmunoEMs from WT retina (FIG. 21A), Myo7a-null retinastreated with 1×AAV2-MYO7A (FIG. 21B) or 1×AAV5-MYO7A (FIG. 21C), andfrom an untreated Myo7a-null retina (FIG. 21D) labeled with anti-rodopsin and 12-nm gold-conjugated secondary antibody. Scale=200 nm;

FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D, and FIG. 22E show a schematicrepresentation of the dual-AAV-vector pairs created for this study. FIG.22A is a fragmented vector (fAAV). FIG. 22B shows simple overlap: the1365-bp shared between the two vectors is shaded gray. FIG. 22C is atrans-splicing vector. FIG. 22D shows an AP hybrid vector: the 270-bpelement shared between the two vectors is marked with diagonal gradientshading (⅓ head as described by Ghosh et al., 2011). FIG. 22 shows thenative intron hybrid vectors utilizing the natural intron 23 of MYO7Asharing a 250-bp overlapping sequence. 3′MYO7A is the 3′-portion ofMYO7A; 5′MYO7A is the 5′-portion of MYO7A; AAV is adeno-associatedvirus; AP is alkaline phosphatase; intron=intron 23 of MYO7A;pA=polyadenylation signal; SA=splice-acceptor site; SD=splice-donorsite; smCBA=cytomegalovirus immediate early/chicken β-actin chimericpromoter;

FIG. 23A, FIG. 23B, and FIG. 23C show human embryonic kidney (HEK293)cells express human MYO7A after infection with simple overlap vectors(MOI of 10,000 for both vectors) packaged in AAV2(tripleY-F). Equalamounts of protein were separated on 7.5% sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and stained for MYO7A. FIG.23A shows HEK293 cells infected with AAV2(tripleY-F) at MOIs of 10,000,2000, and 400 FIG. 23B is a time-course assay of MYO7A expressed inHEK293 cells. Cells were harvested 3-7 days after infection. In FIG.23C, HEK293 cells were infected with AAV2 dual vectors; MOI=multiplicityof infection; T=HEK293 cells transfected with full-length MYO7A plasmid;U=untreated HEK293 cells;

FIG. 24 shows the comparison of AAV2 and AAV2(tripleY→F mutantcapsid)-based vectors in HEK293 cells. Cells were infected with singlefAAV, AP hybrid, and simple overlap MYO7A dual-vector platforms packagedin AAV2 or AAV2(tripleY→F mutant capsid) at an MOI of 10,000. HEK293cells transfected with MYO7A plasmid were used as a positive control;

FIG. 25A, FIG. 25B, and FIG. 25C show human MYO7A expressed in HEK293cells. Cells were infected with AAV2-based vector platforms. For each ofthe dual-vector systems, the corresponding 5′ and 3′ vectors (or the 5′vector alone) were used for infection. HEK293 cells transfected withMYO7A plasmid were used as a positive control. Cells were infected withthe MYO7A dual-vector pairs at an MOI of 10,000 for each vector. Proteinsamples were analyzed on Western blot with an antibody against MYO7A(FIG. 25A). Each platform's relative ability to promote reconstitutionwas compared by quantifying the amount of 5′ vector-mediated truncatedprotein product in the presence or absence of the respective 3′ vector(FIG. 25B). Full-length MYO7A expression mediated by dual vectors wasquantified relative to transfection control (FIG. 25C);

FIG. 26A, FIG. 26B, and FIG. 26C show the characterization of MYO7Adual-vectors' restoration of coding sequence. The experimental plan isshown in FIG. 26A. HEK293 cells were infected with AAV2-baseddual-vector platforms, RNA was extracted, and gene-specific primersamplified the sequences using PCT. Control digests with BglII (B) andPpuMI (P) revealed the predicted banding pattern shown in FIG. 26B.Undigested (U) PCR product is shown as control and a DNA size marker forreference (M). Separately, products were digested with KpnI and AgeI,and then cloned into pUC57 for sequencing of the entire overlap regionFIG. 26C. Ten clones per vector platform were analyzed. M13 forward- andreverse-primers specific for the subclone vector were used to obtainsense and antisense reads (each ˜1000 bp) resulting in ˜440 bp for whichthe sense and antisense reads overlapped. PCR=polymerase chain reactionand

FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, FIG. 27E, FIG. 27F, FIG. 27G,and FIG. 27H show the dual vector-mediated MYO7A(HA) expression in vivo.C57BL/6J mice were injected subretinally with AAV2-based dual vectorscontaining a C′ terminal HA tag. Retinal protein expression was analyzedfour weeks later by immunohistochemistry and western blot. Ten-micronfrozen retinal cross sections were imaged at 10× (FIG. 27A, FIG. 27C,and FIG. 27E) and 60× (FIG. 27B, FIG. 27D, and FIG. 27F). Equal amountsof protein were separated on a 4-15% polyacrylamide gel and stained withan HA antibody (FIG. 27H). For comparison, endogenous MYO7A fromC57BL/6J retina (FIG. 27G) was probed with an antibody against MYO7A toconfirm that HA-tagged MYO7A migrated at the appropriate size.RPE—retinal pigment epithelium, IS—inner segments, OS—outer segments,ONL—outer nuclear layer, INL—inner nuclear layer, GCL—ganglion celllayer, PR—photoreceptors.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of an “hMyo7a coding overlapvector A” of the subject invention;

SEQ ID NO:2 is the nucleotide sequence of an “hMyo7a coding overlapvector B” of the subject invention;

SEQ ID NO:3 is the nucleotide sequence of an “hMyo7a intron 23 splicingvector A” of the subject invention;

SEQ ID NO:4 is the nucleotide sequence of an “hMyo7a intron 23 splicingvector B” of the subject invention;

SEQ ID NO:5 is a nucleotide sequence encoding a human myosin VIIapolypeptide (protein coding sequence is nucleotides 273-6920);

SEQ ID NO:6 is the amino acid sequence of the human myosin VIIapolypeptide encoded by nucleotides 273-6920 of SEQ ID NO:5;

SEQ ID NO:7 is a nucleotide sequence that encodes a human myosin VIIapolypeptide;

SEQ ID NO:8 is an amino acid sequence of a human myosin VIIa polypeptide(isoform 2);

SEQ ID NO:9 is a synthetic oligonucleotide sequence, designated hereinas P1, useful in accordance with one aspect of the present invention;

SEQ ID NO:10 is a synthetic oligonucleotide sequence, designated hereinas P2, useful in accordance with one aspect of the present invention;

SEQ ID NO:11 is a synthetic oligonucleotide sequence, designated hereinas P3, useful in accordance with one aspect of the present invention;

SEQ ID NO:12 is a synthetic oligonucleotide sequence, designated hereinas P4, useful in accordance with one aspect of the present invention;

SEQ ID NO:13 is a synthetic oligonucleotide sequence, designated hereinas P5, useful in accordance with one aspect of the present invention;

SEQ ID NO:14 is a synthetic oligonucleotide sequence, designated hereinas P6, useful in accordance with one aspect of the present invention;

SEQ ID NO:15 is a synthetic oligonucleotide sequence, designated hereinas P7, useful in accordance with one aspect of the present invention;

SEQ ID NO:16 is a synthetic oligonucleotide sequence, designated hereinas P8, useful in accordance with one aspect of the present invention;

SEQ ID NO:17 is a synthetic oligonucleotide sequence, designated hereinas P9, useful in accordance with one aspect of the present invention;

SEQ ID NO:18 is a synthetic oligonucleotide sequence, designated hereinas P10, useful in accordance with one aspect of the present invention;

SEQ ID NO:19 is a synthetic oligonucleotide sequence, designated hereinas P11, useful in accordance with one aspect of the present invention;

SEQ ID NO:20 is a synthetic oligonucleotide sequence, designated hereinas P12, useful in accordance with one aspect of the present invention;

SEQ ID NO:21 is a synthetic oligonucleotide sequence, designated hereinas P13, useful in accordance with one aspect of the present invention;

SEQ ID NO:22 is a synthetic oligonucleotide sequence, designated hereinas P14, useful in accordance with one aspect of the present invention;

SEQ ID NO:23 is a synthetic oligonucleotide sequence, designated hereinas P15, useful in accordance with one aspect of the present invention;

SEQ ID NO:24 is a synthetic oligonucleotide sequence, designated hereinas P16, useful in accordance with one aspect of the present invention;

SEQ ID NO:25 is a synthetic oligonucleotide sequence, designated hereinas P17, useful in accordance with one aspect of the present invention;

SEQ ID NO:26 is a synthetic oligonucleotide sequence, designated hereinas P18, useful in accordance with one aspect of the present invention;

SEQ ID NO:27 is a synthetic oligonucleotide sequence, designated hereinas P19, useful in accordance with one aspect of the present invention;

SEQ ID NO:28 is a synthetic oligonucleotide sequence, designated hereinas P20, useful in accordance with one aspect of the present invention;

SEQ ID NO:29 is a synthetic oligonucleotide sequence, designated hereinas P21, useful in accordance with one aspect of the present invention;and

SEQ ID NO:30 is a synthetic oligonucleotide sequence, designated hereinas P22, useful in accordance with one aspect of the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In theinterest of clarity, not all features of an actual implementation aredescribed in this specification. It will of course be appreciated thatin the development of any such actual embodiment, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which will vary from one implementation toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would be a routine undertakingfor those of ordinary skill in the art having the benefit of thisdisclosure.

The subject invention concerns materials and methods for genetic therapyof diseases and conditions, such as Usher syndrome 1B (USH1B). Oneaspect of the invention concerns AAV-based dual-vector systems thatprovide for expression of full-length proteins whose coding sequenceexceeds the polynucleotide packaging capacity of individual AAV vector.In one embodiment, a vector system of the invention includes:

i) a first AAV vector polynucleotide comprising an inverted terminalrepeat at each end (5′ and 3′ end) of the polynucleotide, and betweenthe inverted terminal repeats a suitable promoter followed by (i.e., 3′to the promoter) a partial coding sequence that encodes an N-terminalpart of a selected full-length polypeptide, and

ii) a second AAV vector polynucleotide comprising an inverted terminalrepeat at each end (i.e., the 5′- and 3′-ends) of the polynucleotide,and between the inverted terminal repeats a partial coding sequence thatencodes a C-terminal portion of the selected full-length polypeptide,and optionally followed by a polyadenylation (pA) sequence. The codingsequences in the first and second vectors when combined encode theselected full-length polypeptide, or a functional fragment or variantthereof. The polypeptide encoding sequence in the first and second AAVvectors comprises sequence that overlaps.

In other words, a portion of the coding sequence present at the 3′-endof the coding sequence of the first vector is identical or substantiallyidentical with a portion of the coding sequence present at the 5′-end ofthe coding sequence of the second vector. In one embodiment, thesequence overlap between the first and second AAV vectors is betweenabout 500 and about 3000 nucleotides; between about 1000 and about 2000nucleotides; between about 1200 and about 1800 nucleotides; or betweenabout 1300 and about 1400 nucleotides.

In a specific embodiment, the sequence overlap is about 1350nucleotides. In an exemplified embodiment, the sequence overlap isapproximately 1365 nucleotides. In one embodiment, the polypeptideencoded is wild type or functional human myosin VIIa (hmyo7a). Aminoacid sequences of wild type and functional hmyo7a polypeptides, andpolynucleotides encoding them, are known in the art (see, for example,GenBank accession numbers NP_(—)000251 and U39226.1). In one embodiment,an hmyo7a polypeptide comprises the amino acid sequence shown in SEQ IDNO:6 or SEQ ID NO:8, or a functional fragment or a variant thereof. Inone embodiment, the hmyo7a polypeptide is encoded by the nucleotidesequence shown in SEQ ID NO:5 or SEQ ID NO:7. Other polypeptidescontemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9),cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), andusherin (USH2A) (Uniprot 075445). In an exemplified embodiment, thefirst AAV vector polynucleotide comprises the nucleotide sequence of SEQID NO:1, or a functional fragment and/or variant thereof, and the secondAAV vector polynucleotide comprises the nucleotide sequence of SEQ IDNO:2, or a functional fragment and/or variant thereof. In oneembodiment, a construct or vector of the invention is administered byparenteral administration, such as intravenous, intramuscular,intraocular, intranasal, etc. In a specific embodiment, a construct orvector is administered by subretinal injection. The construct or vectorcan be administered in vivo or ex vivo.

In another embodiment, a vector system of the invention comprises i) afirst AAV vector polynucleotide comprising an inverted terminal repeatat each end (i.e., the 5′-end and the 3′-end) of the polynucleotide, andbetween the inverted terminal repeats a suitable promoter followed by(i.e., 3′ to the promoter) a partial coding sequence that encodes anN-terminal part of a selected full-length polypeptide followed by asplice donor site and an intron, and ii) a second AAV vectorpolynucleotide comprising an inverted terminal repeat at each end(5′-end and 3′-end) of the polynucleotide, and between the invertedterminal repeats an intron and a splice acceptor site for the intron,followed by a partial coding sequence that encodes a C-terminal part ofthe selected full-length polypeptide, optionally followed by apolyadenylation (pA) signal sequence.

The coding sequences in the first and second vectors when combinedencode the selected full-length polypeptide, or a functional fragment orvariant thereof. The intron sequence in the first and second AAV vectorscomprises sequence that overlaps. In other words, all or part of theintron sequence present at the 3′-end of the coding sequence of thefirst vector is identical or substantially identical with all or part ofthe intron sequence present at the 5′-end of the coding sequence of thesecond vector. In one embodiment, intron sequence overlap between thefirst and second AAV vectors is several hundred nucleotides in length.In a specific embodiment, the intron sequence overlap is about 50 toabout 500 nucleotides or so in length; alternatively between about 200and about 300 nucleotides or so in length. In one embodiment, the intronsequence utilized in the vector system of the invention is a sequence ofan intron naturally present in the genomic sequence of a gene encodingthe selected polypeptide. In one embodiment, the intron is intron 23 ofthe hmyo7a gene. In a specific embodiment, the polypeptide encoded ishmyo7a and the intron is the full intron 23 of the hmyo7a gene. In anexemplified embodiment, the first AAV vector polynucleotide comprisesthe nucleotide sequence of SEQ ID NO:3, or a functional fragment and/orvariant thereof, and the second AAV vector polynucleotide comprises thenucleotide sequence of SEQ ID NO:4, or a functional fragment and/orvariant thereof. In another embodiment, the intron sequence utilized inthe vector system of the invention is a sequence of an intron that isnot naturally present in the genomic sequence of a gene encoding theselected polypeptide. In a specific embodiment, the intron is asynthetic alkaline phosphatase (AP) intron. The intron sequencesutilized in the vector system of the present invention can comprisesplice donor and splice acceptor sequences. In one embodiment, aconstruct or vector of the invention is administered by parenteraladministration, such as intravenous, intramuscular, intraocular,intranasal, etc. In a specific embodiment, a construct or vector isadministered by subretinal injection. The construct or vector can beadministered in vivo or ex vivo.

The inverted terminal repeat (ITR) sequences used in an AAV vectorsystem of the present invention can be any AAV ITR. The ITRs used in anAAV vector can be the same or different. In particular embodiments, theITR may be obtained from an AAV serotype 2 (AAV2) or an AAV serotype 5(AAV5). An AAV vector of the invention can comprise different AAV ITRs.For example, a vector may comprise an ITR of AAV2 and an ITR of AAV5.AAV ITR sequences are well known in the art (see, for example, GenBankAccession Nos. AF043303.1; NC_(—)001401.2; J01901.1; JN898962.1;K01624.1; and K01625.1).

The AAV dual-vector systems disclosed herein are able to efficientlyexpress a therapeutic gene that is larger than what may ordinarily bepackaged within a single AAV vector.

The subject invention also concerns a virus or virion comprising apolynucleotide, expression construct, or vector construct of theinvention. In one embodiment, the virus or virion is an AAV virus.Methods for preparing viruses and virions comprising a heterologouspolynucleotide or construct are known in the art. In the case of AAV,cells can be co-infected or transfected with adenovirus orpolynucleotide constructs comprising adenovirus genes suitable for AAVhelper function. Examples of materials and methods are described, forexample, in U.S. Pat. Nos. 8,137,962 and 6,967,018 (each of which isspecifically incorporated herein, by express reference thereto).

An AAV virus or AAV vector of the invention can be of any AAV serotype,including, but not limited to, serotype AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, and AAV12. In exemplaryembodiments, AAV2 and AAV5 serotype vectors have been utilized.

In one embodiment, the AAV serotype provides for one or more tyrosine tophenylalanine (Y→F) mutations on the capsid surface. In a specificembodiment, the AAV is an AAV8 serotype having atyrosine-to-phenylalanine (Tyr→Phe) mutation at position 733 (Y733F). InFIG. 5, a triple-mutant vector is also contemplated. It containstyrosine-to-phenylalanine (Tyr→Phe) mutations at positions Y733F, Y500F,and Y730F, respectively, corresponding to the amino acid sequence of thewild-type AAV8-capsid protein, or in one or more of the amino acidscorresponding to those sequences in one or more related AAV capids,including, for example, AAV2, AAV5, and the like.

The subject invention also concerns methods for treating or amelioratinga disease or condition, such as an eye disease, in a human or animalusing gene therapy and an AAV-based dual-vector system of the presentinvention. In one embodiment, a method of the invention comprisesadministering a vector system of the invention that encodes apolypeptide that provides for treatment or amelioration of the diseaseor condition. In one embodiment, the vectors of the invention areprovided in an AAV virus or virion. The vector system can beadministered in vivo or ex vivo. In one embodiment, a vector system ofthe invention is administered by parenteral administration, such asintravenous, intramuscular, intraocular, intranasal, etc. Administrationcan be by injection. In a specific embodiment, a vector system of theinvention is administered to the human or animal by intraocularsubretinal injection. In one embodiment, the disease or condition to betreated is Usher syndrome and the polypeptide provided is a mammalianmyosin VIIa protein. In a specific embodiment, the myosin VIIa is ahuman myosin VIIa polypeptide. In one embodiment, an hmyo7a polypeptidecomprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:8,or a functional fragment or a variant thereof. In one embodiment, thehmyo7a polypeptide is encoded by the nucleotide sequence shown in SEQ IDNO:5 or SEQ ID NO:7.

Other polypeptides contemplated include, but are not limited to,harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445). Dosageregimes and effective amounts to be administered can be determined byordinarily skilled clinicians. Administration may be in the form of asingle dose or multiple doses. General methods for performing genetherapy using polynucleotides, expression constructs, and vectors areknown in the art (see, for example, Gene Therapy: Principles andApplications (1999); and U.S. Pat. Nos. 6,461,606; 6,204,251 and6,106,826, each of which is specifically incorporated herein in itsentirety by express reference thereto).

The subject invention also concerns methods for expressing a selectedpolypeptide in a cell. In one embodiment, the method comprisesincorporating in the cell an AAV-based, dual-vector system as disclosedherein, wherein the vector system includes a polynucleotide sequencethat encodes a selected polypeptide and of interest, and expressing thepolynucleotide sequences in the cell. In certain embodiments, theselected polypeptide may be a polypeptide that is heterologous to thecell. In one embodiment, the cell is a mammalian cell, and preferably, ahuman cell. In one embodiment, the cell is human a photoreceptor cell,and preferably a human photoreceptor cone cell or a photoreceptor rodcell. In a specific embodiment, the cell expresses a wild type,functional, and/or biologically-active hmyo7a polypeptide that isencoded by a nucleic acid segment present in a vector system asdisclosed herein. In one embodiment, the hmyo7a polypeptide is encodedby the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:7. Otherpolypeptides contemplated include, but are not limited to, harmonin(Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15(Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445). The cell can beone that is provided in vivo or in vitro.

The subject invention also concerns one or more mammalian cells thatcontain one of the AAV-based, dual-vector systems disclosed herein.

In one embodiment, the cell is a photoreceptor cell. In a specificembodiment, the cell is a cone cell; preferably, it is a human cone cellor a human rod cell. Such cells may express one or more nucleotidesequences provided in at least a first AAV-based, dual-vector system ofthe invention. In a specific embodiment, the cell expresses a wild type,functional, and/or biologically active hmyo7a polypeptide that isencoded by a nucleic acid segment comprised within one or more of theAAV-based vector systems as disclosed herein. In one embodiment, thehmyo7a polypeptide is encoded by the nucleotide sequence of SEQ ID NO:5or SEQ ID NO:7. Other polypeptides contemplated include, but are notlimited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251),protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445).

Vector systems of the invention can include regulatory elements that arefunctional in the intended host cell in which the vector is to beexpressed. A person of ordinary skill in the art can select regulatoryelements for use in appropriate host cells, for example, mammalian orhuman host cells. Regulatory elements include, for example, promoters,transcription termination sequences, translation termination sequences,enhancers, and polyadenylation elements.

A vector of the invention can comprise a promoter sequence operablylinked to a nucleotide sequence encoding a desired polypeptide.Promoters contemplated for use in the subject invention include, but arenot limited to, cytomegalovirus (CMV) promoter, SV40 promoter, Roussarcoma virus (RSV) promoter, chimeric CMV/chicken β-actin promoter(CBA) and the truncated form of CBA (smCBA) (see, e.g., Haire et al.2006 and U.S. Pat. No. 8,298,818, which is specifically incorporatedherein in its entirety by express reference thereto). Additionalphotoreceptor-specific, human rhodopsin kinase (hGRK1) promoter, rodspecific IRBP promoter, VMD2 (vitelliform macular dystrophy/Bestdisease) promoter, and EF1 alpha promoter sequences are alsocontemplated to be useful in the practice of various aspects of thepresent invention.

In a specific embodiment, the promoter is a chimeric CMV-β-actinpromoter. In one embodiment, the promoter is a tissue-specific promoterthat shows selective activity in one or a group of tissues but is lessactive or not active in other tissue. In one embodiment, the promoter isa photoreceptor-specific promoter. In a further embodiment, the promoteris preferably a cone cell-specific promoter or a rod cell-specificpromoter, or any combination thereof. In one embodiment, the promoter isthe promoter for human myosin 7a gene. In a further embodiment, thepromoter comprises a cone transducin a (TaC) gene-derived promoter. In aspecific embodiment, the promoter is a human GNAT2-derived promoter.Other promoters contemplated within the scope of the invention include,without limitation, a rhodopsin promoter, a cGMP-phosphodiesteraseβ-subunit promoter, a retinitis pigmentosa-specific promoter, an RPEcell-specific promoter [such as a vitelliform macular dystrophy-2 (VMD2)promoter (Best1) (Esumi et al., 2004)], or any combination thereof.

Promoters can be incorporated into a vector using standard techniquesknown to those of ordinary skill in the molecular biology and/orvirology arts. Multiple copies of promoters, and/or multiple distinctpromoters can be used in the vectors of the present invention. In onesuch embodiment, a promoter may be positioned about the same distancefrom the transcription start site as it is from the transcription startsite in its natural genetic environment, although some variation in thisdistance is permitted, of course, without a substantial decrease inpromoter activity. In the practice of the invention, one or moretranscription start site(s) are typically included within the disclosedvectors.

The vectors of the present invention may further optionally include oneor more transcription termination sequences, one or more translationtermination sequences, one or more signal peptide sequences, one or moreinternal ribosome entry sites (IRES), and/or one or more enhancerelements, or any combination thereof. Transcription termination regionscan typically be obtained from the 3′ untranslated region of aeukaryotic or viral gene sequence. Transcription termination sequencescan be positioned downstream of a coding sequence to provide forefficient termination. Signal peptide sequences are amino-terminalpeptidic sequences that encode information responsible for the locationof an operably-linked polypeptide to one or more post-translationalcellular destinations, including, for example, specific organellecompartments, or to the sites of protein synthesis and/or activity, andeven to the extracellular environment.

Enhancers—cis-acting regulatory elements that increase genetranscription—may also be included in one of the disclosed AAV-basedvector systems. A variety of enhancer elements are known to those ofordinary skill in the relevant arts, and include, without limitation, aCaMV 35S enhancer element, a cytomegalovirus (CMV) early promoterenhancer element, an SV40 enhancer element, as well as combinationsand/or derivatives thereof. One or more nucleic acid sequences thatdirect or regulate polyadenylation of the mRNA encoded by a structuralgene of interest, may also be optionally included in one or more of thevectors of the present invention.

The disclosed dual-vector systems may be introduced into one or moreselected mammalian cells using any one or more of the methods that areknown to those of ordinary skill in the gene therapy and/or viral arts.Such methods include, without limitation, transfection, microinjection,electroporation, lipofection, cell fusion, and calcium phosphateprecipitation, as well as biolistic methods. In one embodiment, thevectors of the invention may be introduced in vivo, including, forexample, by lipofection (i.e., DNA transfection via liposomes preparedfrom one or more cationic lipids) (see, for example, Feigner et al.,1987). Synthetic cationic lipids (LIPOFECTIN, Invitrogen Corp., LaJolla, Calif., USA) may be used to prepare liposomes that willencapsulate the vectors to facilitate their introduction into one ormore selected cells. A vector system of the invention can also beintroduced in vivo as “naked” DNA using methods known to those ofordinary skill in the art.

Polynucleotides described herein can also be defined in terms of moreparticular identity and/or similarity ranges with those exemplifiedherein. The sequence identity will typically be greater than 60%,preferably greater than 75%, more preferably greater than 80%, even morepreferably greater than 90%, and can be greater than 95%. The identityand/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99% or greater as compared to a sequenceexemplified herein.

Unless otherwise specified, as used herein percent sequence identityand/or similarity of two sequences can be determined using the algorithmof Karlin and Altschul (1990), modified as in Karlin and Altschul(1993). Such an algorithm is incorporated into the NBLAST and XBLASTprograms of Altschul et al. (1990). BLAST searches can be performed withthe NBLAST program, score=100, word-length=12, to obtain sequences withthe desired percent sequence identity. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be used as described (Altschul etal., 1997). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (NBLAST and XBLAST) can be used inaccordance with published methods.

The subject invention also contemplates those polynucleotide moleculeshaving sequences that are sufficiently homologous with thepolynucleotide sequences of the invention to permit hybridization withthat sequence under standard stringent conditions and standard methods(Maniatis et al., 1982). As used herein, “stringent” conditions forhybridization refers to conditions wherein hybridization is typicallycarried out overnight at 20-25° C. below the melting temperature (T_(m))of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, and 0.1% SDS,containing 0.1 mg/mL of a suitable non-specific denatured DNA.Calculation of the melting temperature may be obtained using thestandard formula of Beltz et al., (1983):

T _(m)=81.5° C.+16.6 Log [Na⁺]+0.41(% G+C)−0.61(% formamide)−600/lengthof duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 min in 1×SSPE, 0.1% SDS (i.e., alow-stringency wash); and

(2) Once at T_(m)−20° C. for 15 min in 0.2×SSPE, 0.1% SDS (i.e., amoderate-stringency wash).

As used herein, the terms “nucleic acid” and “polynucleotide sequence”refer to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited, encompassknown analogs of natural nucleotides that can function in a similarmanner as naturally occurring nucleotides. The polynucleotide sequencesinclude both full-length sequences, as well as shorter sequences derivedfrom the full-length sequences. It is understood that a particularpolynucleotide sequence includes the degenerate codons of the nativesequence or sequences that may be introduced to provide codon preferencein a specific host cell. The polynucleotide sequences falling within thescope of the subject invention further include sequences thatspecifically hybridize with the sequences coding for a peptide of theinvention. The polynucleotide includes both the sense and antisensestrands, either as individual strands or in the duplex.

Fragments and variants of a polynucleotide or polypeptide of the presentinvention can be generated as described herein and tested for thepresence of function using standard techniques known in the art. Thus,an ordinarily skilled artisan can readily prepare and test fragments andvariants of a polynucleotide or polypeptide of the invention anddetermine whether the fragment or variant retains functional activitythat is the same or similar to a full-length or a non-variantpolynucleotide or polypeptide, such as a myosin VIIa polynucleotide orpolypeptide.

As those skilled in the art can readily appreciate, there can be anumber of variant sequences of a protein found in nature, in addition tothose variants that can be artificially created by the skilled artisanin the lab. The polynucleotides and polypeptides of the subjectinvention encompasses those specifically exemplified herein, as well asany natural variants thereof, as well as any variants which can becreated artificially, so long as those variants retain the desiredfunctional activity.

Also within the scope of the subject invention are polypeptides whichhave the same amino acid sequences of a polypeptide exemplified hereinexcept for amino acid substitutions, additions, or deletions within thesequence of the polypeptide, as long as these variant polypeptidesretain substantially the same relevant functional activity as thepolypeptides specifically exemplified herein. For example, conservativeamino acid substitutions within a polypeptide that do not affect thefunction of the polypeptide would be within the scope of the subjectinvention. Thus, the polypeptides disclosed herein should be understoodto include variants and fragments, as discussed above, of thespecifically exemplified sequences.

The subject invention further includes nucleotide sequences that encodethe polypeptides disclosed herein. These nucleotide sequences can bereadily constructed by those skilled in the art having the knowledge ofthe protein and amino acid sequences that are presented herein. As wouldbe appreciated by one skilled in the art, the degeneracy of the geneticcode enables the artisan to construct a variety of nucleotide sequencesthat encode a particular polypeptide or protein. The choice of aparticular nucleotide sequence could depend, for example, upon the codonusage of a particular expression system or host cell.

Polypeptides having substitution of amino acids other than thosespecifically exemplified in the subject polypeptides are alsocontemplated within the scope of the present invention. For example,non-natural amino acids can be substituted for the amino acids of apolypeptide of the invention, so long as the polypeptide havingsubstituted amino acids retains substantially the same activity as thepolypeptide in which amino acids have not been substituted. Examples ofnon-natural amino acids include, but are not limited to, ornithine,citrulline, hydroxyproline, homoserine, phenylglycine, taurine,iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid,4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, ε-aminohexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline,cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, C-methyl amino acids, N-methyl aminoacids, and amino acid analogs in general. Non-natural amino acids alsoinclude amino acids having derivatized side groups. Furthermore, any ofthe amino acids in the protein can be of the D- (dextrorotary) or the L-(levorotary) form.

Amino acids can be generally categorized in the following classes:non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a polypeptide having an amino acid of one class isreplaced with another amino acid of the same class fall within the scopeof the subject invention so long as the polypeptide having thesubstitution retains substantially the same biological activity as apolypeptide that does not have the substitution. Table 1 provides alisting of examples of amino acids belonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

Also within the scope of the subject invention are polynucleotides thathave the same, or substantially the same, nucleotide sequence of apolynucleotide exemplified herein, except for the presence of one ormore nucleotide substitutions, additions, or deletions within thesequence of the polynucleotide, so long as these variant polynucleotidesretain substantially the same relevant functional activity as thepolynucleotides exemplified herein (i.e., they encode a protein havingthe same amino acid sequence or the same functional activity as one ofthe polynucleotides specifically exemplified herein). Thus, thepolynucleotides disclosed herein should also be understood to includevariants and fragments thereof.

The methods of the present invention can be used with humans and otheranimals. The other animals contemplated within the scope of theinvention include domesticated, agricultural, or zoo- orcircus-maintained animals. Domesticated animals include, for example,dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs, monkeys orother primates, and gerbils. Agricultural animals include, for example,horses, mules, donkeys, burros, cattle, cows, pigs, sheep, andalligators. Zoo- or circus-maintained animals include, for example,lions, tigers, bears, camels, giraffes, hippopotamuses, andrhinoceroses. As used herein, the terms “patient” and “subject” are usedinterchangeably and are intended to include such human and non-humanspecies. Likewise, in vitro methods of the present invention may also beperformed on cells of one or more human or non-human, mammalian species.

As one of ordinary skill in the molecular biological arts can readilyappreciate, there can be a number of variant sequences of a gene orpolynucleotide found in nature, in addition to those variants that maybe artificially prepared or synthesized by an ordinary-skilled artisanin a laboratory environment. The polynucleotides of the subjectinvention encompasses those specifically exemplified herein, as well asany natural variants thereof, as well as any variants which can becreated artificially, so long as those variants retain the desiredbiological activity.

Also within the scope of the subject invention are polynucleotides whichhave the same nucleotide sequences of a polynucleotide exemplifiedherein except for nucleotide substitutions, additions, or deletionswithin the sequence of the polynucleotide, as long as these variantpolynucleotides retain substantially the same relevant biologicalactivity as the polynucleotides specifically exemplified herein. Thus,the polynucleotides disclosed herein should be understood to includevariants and fragments, as discussed above, of the specificallyexemplified sequences.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

The present invention also concerns pharmaceutical compositionscomprising a vector system of the invention in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor topical or parenteral administration, comprising an amount of acompound constitute a preferred embodiment of the invention. The doseadministered to a patient, particularly a human, in the context of thepresent invention should be sufficient to achieve a therapeutic responsein the patient over a reasonable timeframe, without lethal toxicity, andpreferably causing no more than an acceptable level of side effects ormorbidity. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

The subject invention also concerns kits comprising a vector system ofthe invention in one or more containers. Kits of the invention canoptionally include pharmaceutically acceptable carriers and/or diluents.In one embodiment, a kit of the invention includes one or more othercomponents, adjuncts, or adjuvants as described herein. In oneembodiment, a kit of the invention includes instructions or packagingmaterials that describe how to administer a vector system containedwithin the kit to a selected mammalian recipient. Containers of the kitcan be of any suitable material, e.g., glass, plastic, metal, etc., andof any suitable size, shape, or configuration. In one embodiment, avector system of the invention is provided in the kit as a solid. Inanother embodiment, a vector system of the invention is provided in thekit as a liquid or solution. In certain embodiments, the kits mayinclude one or more ampoules or syringes that contain a vector system ofthe invention in a suitable liquid or solution form.

Multiple distinct AAV-based, dual-vector systems have been created anddisclosed herein for use in gene-replacement therapies, including, forexample, in the treatment of USH1B in human patients. In a specificembodiment, a vector system of the present invention employs twodiscrete AAV vectors that each packages a maximal-size DNA molecule(i.e., ˜4.5 to 4.8 Kb). The two vectors are co-administered to selectedrecipient cells to reconstitute the full-length, biologically-active,Myo7a polypeptide. In these constructs, a portion of overlapping nucleicacid sequence is common to each of the vector genomes (see FIG. 1). Whenco-delivered to suitable cells, the overlapping sequence regionfacilitates the proper concatamerization of the two partial genecassettes. These gene cassettes then undergo homologous recombination toproduce a full-length gene cassette within the cells (see FIG. 1).Shared components of exemplified embodiments of the dual-vector systemsinclude the use of AAV inverted terminal repeats (TR), the small versionof the chimeric CMV/chicken β-actin promoter (smCBA), human Myo7a(hMyo7a) cDNA sequence and the SV40 polyadenylation (pA) signal.

EXEMPLARY DEFINITIONS

In accordance with the present invention, polynucleotides, nucleic acidsegments, nucleic acid sequences, and the like, include, but are notlimited to, DNAs (including, but not limited to, genomic and/orextragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including,but not limited to, rRNAs, mRNAs, and/or tRNAs), nucleosides, as well asone or more nucleic acid segments obtained from natural sources,chemically synthesized, genetically modified, or otherwise prepared orsynthesized in whole or in part by the hand of man.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

The term “effective amount,” as used herein, refers to an amount that iscapable of treating or ameliorating a disease or condition or otherwisecapable of producing an intended therapeutic effect.

The term “operably linked,” as used herein, refers to that the nucleicacid sequences being linked are typically contiguous, or substantiallycontiguous, and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

The term “promoter,” as used herein, refers to a region or regions of anucleic acid sequence that regulates transcription.

The term “regulatory element,” as used herein, refers to a region orregions of a nucleic acid sequence that regulates transcription.Exemplary regulatory elements include, but are not limited to,enhancers, post-transcriptional elements, transcriptional controlsequences, and such like.

The term “substantially corresponds to,” “substantially homologous,” or“substantial identity,” as used herein, denote a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably, atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared.

The percentage of sequence identity may be calculated over the entirelength of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides.

When highly-homologous fragments are desired, the extent of percentidentity between the two sequences will be at least about 80%,preferably at least about 85%, and more preferably about 90% or 95% orhigher, as readily determined by one or more of the sequence comparisonalgorithms well-known to those of ordinary skill in the art, such ase.g., the FASTA program analysis described by Pearson and Lipman (1988).

The term “subject,” as used herein, describes an organism, includingmammals such as primates, to which treatment with the compositionsaccording to the present invention can be provided. Mammalian speciesthat can benefit from the disclosed methods of treatment include, butare not limited to, humans, non-human primates such as apes;chimpanzees; monkeys, and orangutans, domesticated animals, includingdogs and cats, as well as livestock such as horses, cattle, pigs, sheep,and goats, or other mammalian species including, without limitation,mice, rats, guinea pigs, rabbits, hamsters, and the like.

The term “treatment” or any grammatical variation thereof (e.g., treat,treating, and treatment etc.), as used herein, includes but is notlimited to, alleviating a symptom of a disease or condition; and/orreducing, suppressing, inhibiting, lessening, ameliorating or affectingthe progression, severity, and/or scope of a disease or condition.

The term “vector,” as used herein, refers to a nucleic acid molecule(typically one containing DNA) that is capable of replication in asuitable host cell, or one to which another nucleic acid segment can beoperatively linked so as to facilitate replication of theoperably-nucleic acid segment. Exemplary vectors include, withoutlimitation, plasmids, cosmids, viruses and the like.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Dual-AAV Vector System 1: hMyo7a Coding Overlap

In the case of Dual-Vector System 1 (FIG. 2) the overlapping DNAsequence shared by both vector A and vector B consists of a 1350-bpcoding region for the human Myo7a gene. This is the simplest system ofthe present invention, and appears to be highly efficient in terms offull-length gene reconstitution, and MYO7A expression. Advantageously,each vector is of standard AAV packaging size, and as such, eachpackages DNA with a high degree of efficiency, and is readily adaptableto conventional GMP standards. Such vectors are also readilycharacterized to permit requisite regulatory approval prior to use inhumans.

Example 2 Dual-AAV Vector System 2: hMyo7a Intron 23 Splicing

In the case of Dual-Vector System 2 (FIG. 3) the overlapping DNAsequence is composed of the native intron 23 of human Myo7a. Vector Acontains the coding sequence corresponding to the amino-terminal portionof the hMyo7a cDNA relative to intron 23 (hMyo7aNT) and the nativesplice-donor site, followed by the entire intron 23 of hMyo7a (minus thenative acceptor site). Vector B contains the carboxy-terminal portion ofthe hMyo7a cDNA relative to intron 23 (hMyo7aCT), and the full intron 23of Myo7a (minus the native splice-donor site), followed by the nativesplice-acceptor site. Upon co-delivery to suitable mammalian host cells,the DNA of vectors A and B recombine to form a reconstituted full-lengthgene cassette. The resulting RNA transcript will then ‘splice out’ thenative intron. Alternatively, recombination and formation of the genecassette can occur via the AAV TRs. In this case, the RNA transcriptwill ‘splice out’ the native intron23-TR-intron23 motif. In both cases,however, the resulting mRNA is that of the reconstituted full-lengthhMyo7a gene sequence.

Example 3 In Vitro Performance of System 1: hMyo7a Coding Overlap

HEK293 cells were infected simultaneously with vector A and vector B ofDual-Vector System 1 (FIG. 2) at a ratio of 10000:1 vg/cell for eachvector. The AAV vectors were then packaged in AAV2 virions that containthree Y→F mutations in the capsid protein (see, for example, Zhong etal., 2008). As a positive control, cells were transfected with plasmidcontaining full-length hMyo7a under the control of smCBA. Protein wasrecovered from cells at 3-, 4-, 5-, 6- and 7-days' post-infection, andan antibody directed against MYO7A was used to assay for its presence inthe infected cells via immunoblotting. The results are shown in FIG. 6Aand FIG. 6B. The area inside the white box is magnified, and presentedat higher contrast on the right. Starting at 3-days' post-infection, thefull-length human MYO7a protein was visible; peak expression of theprotein occurred around Day 5.

Example 4 In Vivo Performance of System 1: hMyo7a Coding Overlap

Six week old shaker-1 (Myo7a null) mice were sub-retinally co-injectedwith 1 μL of the same preparations of vector A and vector B ofDual-Vector System 1 used in the above in vitro study. Both vectorscontained ˜1×10¹² vg/mL. Four weeks' post-injection, retinas fromtreated and untreated eyes were collected and immunohistochemistry (IHC)was performed using an antibody directed against MYO7A (see FIG. 7A andFIG. 7B). The area in green showed MYO7A-specific staining, while theareas in blue corresponded to the nuclear-specific, DAPI stain. In thetreated eye, MYO7A expression was clearly visible, and it appeared to berestricted to photoreceptors—more precisely to the juncture of thephotoreceptor inner and outer segments.

Example 5 AAV Dual Vectors Efficiently Deliver Oversized Genes

Animals.

Shaker-1 mice carrying the 4626SB allele, an effective null mutation(Liu et al., 1999; Hasson et al., 1997), were used on the C57BL6 geneticbackground, and maintained and genotyped as described (Liu et al., 1999;Gibbs et al., 2003a). They were maintained on a 12-hr light/12-hr darkcycle, with exposure to 10-50 lux of fluorescent lighting during thelight phase, and were treated according to federal and institutionalanimal care guidelines. Homozygous mutants were distinguished from theheterozygous controls by their hyperactivity, head-tossing and circlingbehavior (Gibson et al., 1995), and/or by a PCR/restriction digestassay.

Construction of AAV Vectors.

Single-vector platform: AAV vector plasmid, containing the truncatedchimeric CMV/chicken β-actin promoter (smCBA) (Haire et al., 2006) andMYO7A cDNA was constructed by removing the full MYO7A cDNA from pEGFP-C2by EagI and SalI digest, and then ligating into pTR-smCBA-GFP that hadbeen digested with NotI and SalI to remove GFP. The MYO7A cDNA (˜6.7 kb)corresponded to isoform 2 of human MYO7A, and was the same as thatdescribed previously by Hashimoto et al. (2007), which was based on thesequence published by Chen et al. (1996) (see, SEQ ID NO:8). MYO7Aisoform 2 is 114-kb shorter than isoform 1 (Chen et al., 1996; Weil etal., 1996). Both the MYO7A cDNA, and the resulting junctions were fullysequenced prior to packaging. All vectors intended for in vitro analyseswere separately packaged in wild type AAV2, or alternatively in theAAV2(tripleY→F) capsid mutant vector (Petrs-Silva et al., 2011). Asnoted above, AAV2-based vectors were chosen for the in vitro experimentsdue to their increased transduction efficiency relative to otherserotypes (Ryals et al., 2011). All vectors were packaged, purified, andtitered using standard methods as previously described (Zolotukhin etal., 2002; Jacobson et al., 2006). Human embryonic kidney (HEK293) cellswere transfected by the calcium phosphate method with vector plasmidcarrying the full-length MYO7A coding sequence of variant 2 (the plasmidused to package fAAV). These transfected cells were then used as apositive control throughout immunoblot analyses to indicate theappropriate size of full-length MYO7A protein. Vector infections werecarried out in HEK293 cells with titer-matched AAV vectors. In brief,cells were grown to 60-70% confluency. All vectors were diluted in abalanced salt solution to achieve the desired multiplicity of infection(MOI). If not specifically mentioned, cells were infected at 10,000genome-containing particles/cell of each vector, resulting in an MOI of20,000 total for each vector pair. Cells were incubated in mediumcontaining 10% serum for 3 days post-infection at 37° C. under 7% CO₂,and then analyzed via immunoblot. Titers of 10¹² to 10¹³ particles/mLwere obtained for different lots of AAV2-MYO7A and AAV5-MYO7A.

Oligonucleotide Sequences.

For in vivo studies, a human influenza hemagglutinin (HA) tag was addedto the 3′ termini of the full-length, simple overlap, trans-splicing,and hybrid 3′ vectors by utilizing a unique BamHI site (P19), andreplacing the non-tagged 3′-end with an HA-tagged (P20) version. Allconstructs were sequence verified by Sanger sequencing.

AAV Vector Plasmid Design and Cloning.

The full-length coding sequence of MYO7A (human isoform 2; GenBankAccession No. NM_(—)001127180) was cloned into a vector plasmidcontaining the strong, ubiquitous CMV/chicken β-actin (smCBA) promoter(Haire et al., 2006), a polyadenylation signal, and the AAV2 ITRs.Packaging of this plasmid generated the fAAV vector (FIG. 22A). In allsystems, the 5′ vectors shared the smCBA promoter and a 5′ portion ofMYO7A, whereas the 3′ vectors contained a 3′-portion of MYO7A, and abovine growth hormone (bGH) polyadenylation signal. Oligonucleotidesused for vector construction are listed in Table 2. The simple overlapcontained nucleotides 1 through 3644 of MYO7A cDNA from the ATG in the5′ vector, and nucleotides 2279 through 6534 in the 3′ vector. Thefragments were amplified with oligonucleotides P1 and P3 by polymerasechain reaction (PCR) and cloned into the 5′ vector via NotI and NheI,and the 3′ vector with P3 (AflII) and P4 (KpnI), respectively. Theresulting two vector plasmids share 1365 bp of overlapping MYO7Asequence (FIG. 22B). The trans-splicing and hybrid vectors utilizesplice junctions composed of either ideal splice donor and acceptorsites derived from AP coding sequence or native MYO7A splice junctionsfrom exons 23 and 24 (Yan et al., 2002). To create the 5′ trans-splicingvector, the splice-acceptor site was amplified using oligonucleotides P5and P6 (NheI), and the amplicon was then used in a second reaction witholigonucleotide P7 (NsiI) to add a part of the MYO7A coding sequence forcloning. The corresponding 3′ vector was similarly created by amplifyingthe splice-acceptor site with oligonucleotides P8 (AflII) and P9 in afirst PCR, and adding part of the 3′ MYO7A coding sequence witholigonucleotide P10 (AgeI) in a second PCR (see FIG. 22C). The AP hybridvectors were created by adding 270 bp of AP overlap sequence to therespective trans-splicing vectors (Ghosh et al., 2011). The sequence wasamplified by PCR and, in so doing, appropriate restriction endonucleasesites were added. For the 5′ vector oligonucleotides P11 (NheI) and P12(SalI) were used, while oligonucleotides P13 (NotI) and P14 (AflII) wereused for the 3′ vector (FIG. 22D). A fourth vector pair, “native intronhybrid” vector, was also created to exploit the natural sequence in andaround intron 23 of MYO7A as a recombination locus, and subsequentsplicing signal. The 5′-portion was created by amplifying intron 23 witholigonucleotides P15 and P16 (NheI) first, and then using the resultingamplicon in a second reaction with oligonucleotide P7 (NsiI) tofacilitate cloning. The corresponding 3′-vector was constructed byamplifying the intron 23 with oligonucleotides P17 and P18 (AflII), andthe resulting amplicon, with oligonucleotide P10 (AgeI) in a secondreaction (see FIG. 22E).

TABLE 2 OLIGONUCLEOTIDES USED IN THIS STUDY Restriction Oligo 5′-3′sequence (restriction sites underlined) site (SEQ ID NO:) P1GCGGCGGCCGCCACCATGGTGATTCTTCAGCAGGGGGAC NotI (SEQ ID NO: 9) P2GCGGCTAGCGAAGTTCCGCAGGTACTTGAC NheI (SEQ ID NO: 10) P3GCGCTTAAGCAGGTCTAACTTTCTGAAGCTG AflII (SEQ ID NO: 11) P4GCGGGTACCTCACTTGCCGCTCCTGGAGCC KpnI (SEQ ID NO: 12) P5GGCACCTAGTGGCTTTGAGGTAAGTATCAAGGTTACAAGAC (SEQ ID NO: 13) P6GCGGCTAGCTCAGAAACGCAAGAGTCTTC NheI (SEQ ID NO: 14) P7CTTCTTTGTGCGATGCATCAAG NsiI (SEQ ID NO: 15) P8GCGCTTAAGCGACGCATGCTCGCGATAG AflII (SEQ ID NO: 16) P9CGCCCTCGCTCCAGGTCCTGTGGAGAGAAAGGCAAAG (SEQ ID NO: 17) P10GAACCCGAACCGGTCCTTG AgeI (SEQ ID NO: 18) P11 GCGGCTAGCCCCCGGGTGCGCGGCGNheI (SEQ ID NO: 19) P12 GCGGTCGACGAAACGGTCCAGGCTATGTG SalI(SEQ ID NO: 20) P13 GCGGCGGCCGCCCCCGGGTGCGCGGCG NotI (SEQ ID NO: 21) P14GCGCTTAAGGAAACGGTCCAGGCTATGTG AflII (SEQ ID NO: 22) P15CAGGCACCTAGTGGCTTTGAGGTACCAGGCTAGGGACAGG (SEQ ID NO: 23) P16GCGGCTAGCCGCCTGAGCCCAGAAGTTC NheI (SEQ ID NO: 24) P17CGCCCTCGCTCCAGGTCCTGAAGGAGACAAGAGGTATG (SEQ ID NO: 25) P18GCGCTTAAGCACCGCTTGTGTTGATCCTC AflII (SEQ ID NO: 26) P19GCCAGGGAAGGATCCCATG BamHI (SEQ ID NO: 27) P20 GCGGGTACCTCATGCGTAATCCGGTACATCGTAAGGGTACTTGCCGCTCCTGGAGCC KpnI (SEQ ID NO: 28)P21 AGCTTCGTAGAGTTTGTGGAGCGG (SEQ ID NO: 29) P22 GAGGGGCAAACAACAGATG(SEQ ID NO: 30) Oligonucleotides were used to make 5′ and 3′ vectors ofthe dual-vector platforms (P1-P20). Oligonucleotides were used tocharacterize the fidelity of the overlap in simple overlap,trans-splicing and AP hybrid vector platforms (P21-P22). Restrictionsites used for cloning are underlined and the introduced HA tag is notedin italics (P19).

Dual-Vector Platform.

Two separate vector plasmids were constructed: Vector A contains thestrong, ubiquitous “smCBA” promoter and MYO7A cDNA encoding theN-terminal portion. Vector B contains MYO7A cDNA encoding the C-terminalportion and a poly-A signal sequence. Each vector plasmid contained bothinverted terminal repeats (ITRs). Using PCR with full-length MYO7A cDNAas a template, the MYO7A cDNA was divided roughly in half with ampliconsencompassing nucleotide positions 1 through 3644 (Vector A) and 2279through 6647 (Vector B) relative to ATG start position 1. The resultingtwo-vector plasmids shared 1365 bp of overlapping MYO7A sequence, andwere 5.0- and 4.9-Kb in length, respectively. This was well within thesize limitation of standard AAV vectors. Both vector plasmids weresequence verified and separately packaged by standard AAV productionmethods (Zolotukhin et al., 2002; Jacobson et al., 2006). The titer ofthe first lot contained 2.5×10¹² particles/mL of each vector, and thesecond lot contained 4×10¹² particles/mL of each vector.

Reverse Transcription and Characterization of Overlap Region.

HEK293 cells were infected with dual vectors, and total RNA wasextracted with the RNeasy® kit (Qiagen, Hilden, Germany) according tothe manufacturer's recommended protocol. Two micrograms of RNA was thensubjected to DNaseI (NEB) digestion for 30 min at 37° C., followed byheat inactivation at 75° C. for 10 min Reverse transcription to cDNA wasachieved with the SuperscriptIII® kit (Life Technologies, Grand Island,N.Y., USA) according to the standard protocol utilizing the oligo dTprimer. Two microliters of cDNA was used as template in a PCR (95° C.for 3 min initial denature, 35 cycles of 95° C. for 45 sec, 55° C. for45 sec, 72° C. for 12 min, and a final 72° C. for 15 min) usingoligonucleotide primers P21 and P22 (see Table 2). Annealing sites forthese primers are located 5′ and 3′, respectively, of the area of cDNAoverlap (in other words, outside the region of overlap) in the simpleoverlap and hybrid vector pairs. The 3′-primer annealed to sequence thatwas complimentary to the bGH polyA. Resulting products were digestedwith either PpuMI or BglII, separated on a 1.5% agarose gel, andsubsequently analyzed on a UV screen. Separately, products were digestedwith KpnI and AgeI, and subsequently cloned into a pUC vector forsequencing of the entire overlap region. M13 forward and reverse primersthat were specific for the vector were used to obtain sense andantisense reads resulting in an ˜140 bp overlap of the sense andantisense reads. To demonstrate that these methods were capable ofdetecting aberrant sequence (i.e., quality control), a MYO7A sequencewas generated using either an artificial insertion (HindIII fill-in atposition 2635) or a point mutation (T→C) at position 2381, and theanalyses were repeated.

Viral Delivery In Vitro.

HEK293A cells (Invitrogen), grown in DMEM with 10% FBS and 1×NEAA andPen/Strep (Invitrogen) were plated in 6 well-plates. The next day cellswere incubated, at 37° C. and 5% CO₂, with AAV2- and AAV5-MYO7A at anMOI of 10,000 viral particles/cell in 500 μL of complete medium,containing also 40 μM of calpain inhibitor (Roche, Pleasanton, Calif.,USA). Two hours later complete medium was added. The next day, themedium was changed and cells were incubated for an additional 48 hrs.Alternatively, some cells were transfected with 1 μg of vectorpTR-smCBA-MYO7A, complexed with Lipofectamine 2000 (ratio 1:3),according to the manufacturer's instructions (Invitrogen).

Primary mouse RPE cells were derived from P14-P16 Myo7a-null animals andcultured in 24-well dishes, as described (Gibbs et al., 2003a; Gibbs andWilliams, 2003b). After 48 hrs in culture, cells were transduced withviruses. Cells were incubated in 100 μL of complete medium containing 40μM of calpain inhibitor, and 10,000 viral particles/cell fromfull-strength AAV stocks. After 2 hrs, 400 μL of complete medium wasadded to each well, and incubated overnight. The medium was changed thefollowing day, and cells were incubated for an additional 48 hrs.

ARPE19 cells (American Type Culture Collection, Manassas, Va., USA) werecultivated in DMEM/F-12 with 10% FBS and split into 24-well plates withglass coverslips. Cells were grown to confluency and then transduced inthe same manner, as were the primary RPE cells.

MYO7A Expression Analysis by Western Blot and Immunofluorescence.

HEK293A and primary mouse RPE cells that were transduced with AAV-MYO7Awere collected 3 days post-transduction. For western blot analyses,cells were collected and lysed in 20 mM TRIS, pH 7.4, 5 mM MgCl₂, 10 mMNaCl, 1 mM DTT and 1× protease inhibitor cocktail (Sigma-AldrichChemical Co., St. Louis, Mo., USA). Equivalent amounts of total proteinwere separated on a 7.5% SDS-PAGE gel. After transfer, blots wereblocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody,generated against residues 927-1203 of human MYO7A (DevelopmentalStudies Hybridoma Bank, Iowa City, Iowa USA) (Soni et al., 2005), andmouse anti-actin antibody (Sigma-Aldrich) as a loading control.

Immunofluorescence was performed with ARPE19 and mouse RPE primarycells, 3 days after infection. Cells were fixed in 4% formaldehyde,blocked with blocking solution (0.5% BSA/0.05% saponin in PBS),incubated with the mouse anti-MYO7A followed by goat anti-mouseAlexa-568 (Molecular Probes, Carlsbad, Calif., USA). Coverslips weremounted with mounting medium containing DAPI (Fluorogel II, ElectronMicroscopy Sciences, Hatfield, Pa., USA) and visualized on a Leicaconfocal system.

Protein Extraction and Immunoblotting.

Transfected and infected HEK293 cells were harvested and washed twice inPBS and processed as previously reported with minor modifications (Boyeet al., 2012). The cells were lysed by 3×30 sec pulses of sonication in200 μL of sucrose buffer (0.23 M sucrose, 2 mM EDTA, 5 mM Tris-HCl, pH7.5) containing protease inhibitors (Roche, Mannheim, Germany). Unlysedcells and cell debris were removed by centrifugation at 14,000 rpm for10 min. The protein concentration of the supernatant was measured withBCA (Thermo Fisher Scientific, Rockland, Ill., USA). Equal amounts ofprotein were then loaded on 7.5% sodium dodecyl sulfate polyacrylamidegel electrophoresis gels (BioRad, Hercules, Calif., USA) and transferredin CAPS buffer (pH 11) onto PVDF membranes (Millipore, Billerica,Mass.). Blots were then labeled with antibodies against MYO7A(monoclonal antibody raised against amino acids 11-70 of human MYO7A;Santa Cruz, Dallas, Tex., USA; 1:1000) or HA (MMS-101P; Covance,Gaithersburg, Md., USA; 1:500) and β-actin (ab 34731; Abcam, Cambridge,Mass., USA; 1:5000). For visualization with the Odyssey system (Li-Cor,Lincoln, Nebr., USA), an antimouse and an anti-rabbit secondary antibodyconjugated with CW800 and IR680 dyes (Li-Cor), respectively, were used.Semiquatitative densitometric measurements were performed with Odysseyacquisition and analysis software (Li-Cor). The dual-color images wereseparated in their respective channels and converted to gray scale forpresentation purposes. Size markers present in one channel of each blotwere added to both channels for visualization of protein sizes.

Viral Delivery In Vivo.

Mice were anesthetized with 2.0-3.0% isoflurane inhalation. The pupilsof the animals were dilated with 1% (wt./vol.) atropine sulfate and 2.5%phenylephrine. A local anesthetic (0.5% proparacaine hydrochloride) wasalso administered. A sclerotomy in the temporal limbus was performedwith a 27-Ga needle. A 32-Ga blunt needle, attached to a microsyringepump (WPI, Sarasota Fla., USA) was inserted and 1 μL of viral solutionwas injected into the ventral subretinal space of P14-P16 animals.Retinal detachment was visualized under a dissecting microscope, andregistered as indication of a positive subretinal injection. Onemicroliter of the following AAV8(Y733F)-based vectors was injectedsubretinally in one eye of C57BL/6 mice: single fAAV (1×10¹³ vg/mL),front and back half “hybrid” vectors combined equally (eachvector=1×10¹³ vg/mL), or front and back half “simple overlap” vectorscombined equally (each vector=1×10¹³ vg/mL). Subretinal injections wereperformed as previously described (Timmers et al., 2001). Furtheranalysis was carried out only on animals that received comparable,successful injections (>60% retinal detachment with minimal surgicalcomplications).

Light Microscopy and Immunoelectron Microscopy of Retinas.

Eyecups were processed for embedment in either LR White or Epon, andsemithin and ultrathin sections were prepared. Semithin sections werestained with toluidine blue and visualized on a Leica confocal system.Ultrathin sections were labeled with purified MYO7A pAb 2.2 (Liu et al.,1997) and monoclonal anti-opsin (1D4, R. Molday), followed bygold-conjugated secondary antibodies (Electron Microscopy Sciences), asdescribed previously (Lopes et al., 2011). Negative control sectionsprocessed at the same time included those from Myo7a-null retinas, and,as positive control, WT animals were used.

MYO7A immunogold density was determined on sections of age-matched WT,Myo7a-null retinas and retinas of Myo7a-null animals that had beeninjected with AAV-MYO7A at P14-16 and dissected three weeks later. Forquantification of the immunolabel, all of the gold particles in acomplete section of each RPE cell were counted. The area of each cell'sprofile was determined using ImageJ software. For background labeling,the concentration of label in sections of untreated Myo7a-null animalswas measured. Data were expressed with this background labelingsubtracted.

The concentration of MYO7A and opsin immunogold labeling in theconnecting cilia of photoreceptor cells was determined by counting goldparticles along longitudinal profiles of connecting cilia and measuringthe length of each profile.

Analysis and quantifications were performed in a minimum of threedifferent retinas, from three different animals. Statistical analysiswas performed using one-tail Student's t-test.

Six weeks postinjection, C57BL/6 mice were enucleated and their eyesprocessed and immunostained as previously described (Boye et al., 2011)with minor modifications. Retinas were immunostained with an antibodyspecific for HA (monoclonal Ab clone 12CA5; Roche), counterstained withDAPI, and imaged with a spinning disk confocal microscope (Nikon EclipseTE2000 microscope equipped with Perkin Elmer Ultraview Modular LaserSystem and Hamamatsu O-RCA-R2 camera). Images were obtained sequentiallyusing a 20×(air) objective lens. All settings (exposure, gain, laserpower) were identical across images. All image analysis was performedusing Volocity 5.5 software (Perkin Elmer, Waltham, Mass., USA).

Results

AAV-MYO7A Single Vector Preparations.

AAV vector plasmid was engineered to contain a truncated chimericCMV/chicken β-actin promoter, smCBA (Haire et al., 2006) and the 6.7-kbcDNA encoding the full-length isoform 2 of human MYO7A (NCBI #NM_(—)001127180) (FIG. 10A). The smCBA promoter exhibits the sametropism and activity in mouse retinas as that of the full-length CBApromoter (Haire et al., 2006; Pang et al., 2008). Titers of 10¹² to 10¹³particles/mL were obtained for different lots of AAV2-MYO7A andAAV5-MYO7A. A concentration of 10¹² particles/mL was regarded as thestandard concentration (1×), from which dilutions were made. Theexperiments were performed with virus obtained from three separatepreparations. No differences in expression or phenotype correction, asdescribed below, were observed among the different lots for AAV2-MYO7Aor AAV5-MYO7A at a given concentration.

MYO7A Expression in Cell Culture.

Transduction of primary cultures of Myo7a-null RPE cells with 1× singleAAV2-MYO7A or AAV5-MYO7A resulted in the expression of a polypeptidethat, by western blot analysis, had an apparent mass that was comparableto that of WT MYO7A protein, and was present at similar levels to thatfound in primary cultures of Myo7a^(+/−) RPE cells (FIG. 10B). Likewise,a single band of appropriate size was detected on western blots ofHEK293A cells. Immunofluorescence of the primary RPE cells showed thatthe MYO7A protein, resulting from 1× single AAV-MYO7A treatment ofMYO7A-null cells, had a subcellular localization pattern that wascomparable to that of endogenous MYO7A in control cells, indicating thegeneration of appropriately targeted protein (FIG. 10C, FIG. 10D, FIG.10E, and FIG. 10F). ARPE19 cells were also infected with 1× or diluted(1:100) AAV2-MYO7A or AAV5-MYO7A, and compared with non-treated cells.An increase in MYO7A immunofluorescence was detected in the treatedcells, and the intracellular localization of the label was comparable tothat in untreated cells (FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG.17E, and FIG. 17F).

Localization of MYO7A In Vivo.

Most retinal MYO7A is found in the RPE (Hasson et al., 1995), however,the protein is also present in the connecting cilium and pericilium ofthe photoreceptor cells (Liu et al., 1997; Williams, 2008). A diagramillustrating this distribution and the retinal functions of MYO7A hasbeen published in a recent review (Williams and Lopes, 2011).

Three weeks following injection of 1×AAV2-MYO7A or AAV5-MYO7A into thesubretinal space of Myo7a-null mice, retinal tissue was examined byimmunoelectron microscopy to test for MYO7A expression Immunogold labelwas evident in the photoreceptor cells, where it was localized in theconnecting cilium and pericilium, comparable to that in WT retinas (FIG.11A, FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E). Label was also presentthroughout the RPE cells, particularly in the apical cell body region(FIG. 11F, FIG. 11F-1, FIG. 11G, and FIG. 11G-1; see FIG. 18A, FIG. 18B,FIG. 18C, and FIG. 18D for controls), as found in WT retinas (Gibbs etal., 2004; Liu et al., 1997).

MYO7A has a similar distribution in both rod and cone photoreceptorcells (Liu et al., 1999). To test whether treatment with AAV-MYO7A alsoaffected cone photoreceptor cells, it was determined whether MYO7A wasalso present in the ciliary region of cone photoreceptors. DoubleimmunoEM of treated retinas was performed, using a MYO7A antibodytogether with an antibody specific for rod opsin. Although there areonly a small number of cones with aligned connecting cilia found in eachultrathin section, MYO7A immunogold label was evident in the connectingcilium and periciliary region of these cones, which were identified bylack of rod opsin labeling in their outer segments (in contrast to thesurrounding rod outer segments) (FIG. 11H and FIG. 11I). Hence,AAV2-MYO7A and AAV5-MYO7A can transduce cone as well as rodphotoreceptor cells.

Dose-Dependent MYO7A Expression in Photoreceptor and RPE Cells.

To determine the levels of MYO7A expression following treatment withdifferent concentrations of AAV2-MYO7A and AAV5-MYO7A (1×, 1:10 or 1:100dilutions), MYO7A immunogold labeling was quantified in EM images, takenwithin 1.4 mm of the injection site. Reliable detection of MYO7A in thephotoreceptor cells, where its distribution is limited to the connectingcilium and pericilium, requires the higher resolution provided byelectron microscopy (Liu et al., 1997). Immunogold particle density wasmeasured in images of the photoreceptor connecting cilium andpericilium, shown in complete longitudinal section (from the basalbodies to the base of the outer segment), and in images showing the RPEcells in apical to basal section. Particle density was expressed asparticles per length of cilium for the photoreceptor cells (eachconnecting cilium is ˜1.2 μm long), and as particles per area for theRPE cells (the entire area between the apical and basal surfaces wasincluded). Particle density is dependent on exposure of epitopes on thesurface of the section, and, as such, provides a relative linear measureof antigen density under the conditions used here (i.e., grids wereetched and labeled in an identical manner, and the labeling was not sodense as to be affected by steric hindrance).

Treatment with 1×AAV2-MYO7A or AAV5-MYO7A resulted in 2.5-2.7 times thedensity of immunolabel in the photoreceptor cilium, compared with thatfound in WT retinas, while the 1:10 and 1:100 dilutions resulted in adensity of immunolabel that was more comparable to WT levels (FIG. 11J,FIG. 11L, and FIG. 19). Quantification of immunogold label in the RPEshowed that injection of AAV2-MYO7A resulted in 2.7 times more labelthan in WT, with the 1:10 and 1:100 dilutions showing no significantdifference (FIG. 11K). In contrast, the level of MYO7A immunolabel inthe RPE of retinas injected with AAV5-MYO7A varied in relation to virustiter, with the full dose virus effecting 2.2-fold more MYO7A than thatfound in WT RPE, the 1:10 dilution effecting WT levels, and the 1:100dilution resulting, on average, ˜60% of WT levels (FIG. 11M).

These counts of labeling density indicate that 1×AAV-MYO7A resulted inmore than double the normal level of MYO7A expression in both thephotoreceptor and RPE cells. The distribution of MYO7A was not affectedby this overexpression in the photoreceptor cells. In the RPE cells, theoverall distribution of MYO7A was comparable to WT, with a higherconcentration in the apical cell body region. However, with 1×AAV2-MYO7Aor 1×AAV5-MYO7A, the proportion of MYO7A that was associated withmelanosomes was only 55% of that in WT RPE: This difference is possiblybecause the proteins that link MYO7A to the melanosomes, MYRIP andRAB27A (Klomp et al., 2007; Lopes et al., 2007), may have remained nearWT levels, and thus limited the absolute amount of MYO7A that couldassociate with the melanosomes.

Despite the overexpression of MYO7A, no pathology was evident inretinas, up to 3 months after injection of 1× (or 1:10) AAV2-MYO7A.However, two out of six retinas injected with 10¹³ particles/mL ofAAV5-MYO7A (i.e., 10×) showed evidence of photoreceptor cell loss acrossthe retina after 3 weeks (AAV2-MYO7A was not tested at this titer) (FIG.19).

Correction of Melanosome Localization in the RPE.

In Myo7a-mutant mice, melanosomes are absent from the apical processesof the RPE cells (Liu et al., 1998). This mutant phenotype is evident atall neonatal ages, and is due to loss of actin-based transport of themelanosomes by the myosin 7a motor (Gibbs et al., 2004). Three weeksfollowing injection of 1×AAV2-MYO7A or AAV5-MYO7A into the subretinalspace of Myo7a-null mice, melanosomes were observed to have a normaldistribution in all RPE cells near the site of injection (within 1.4 mm)(n=10 each for AAV2-MYO7A and AAV5-MYO7A) (FIG. 12A, FIG. 12B, and FIG.12C). Well away from the injection site, a mixture of corrected anduncorrected RPE cells was evident, while, at the periphery of theretina, the cells all exhibited the Myo7a-mutant phenotype, indicatinglack of correction in this region (FIG. 12D, FIG. 12E, and FIG. 12F).The correction of melanosomes was still evident in retinas that werefixed 3 months after injection (FIG. 20). Correction was also observedin all eyes injected with 1:10 dilution AAV2-MYO7A (n=6) or AAV5-MYO7A(n=6), as well as in all eyes injected with 1:100 dilution AAV2-MYO7A(n=6) or AAV5-MYO7A (n=6), although with the 1:100 dilution some of theRPE cells near the site of injection were not corrected.

Correction of Opsin Distribution.

Myo7a-mutant mice have an abnormal accumulation of opsin in theconnecting cilia of the photoreceptor cells, a phenotype that is evidentby immunoEM with opsin antibodies (Liu et al., 1999). This mutantphenotype suggested that myosin 7a functions in the vectorial deliveryof opsin to the outer segment (Liu et al., 1999). Quantification ofimmunogold opsin labeling in the connecting cilia, demonstrated thatthis phenotype was corrected with 1×AAV2-MYO7A or AAV5-MYO7A (FIG. 13;FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D). This analysis also showedphenotype correction with 1:100 dilutions, although the data indicatedthat a full WT phenotype was not achieved (FIG. 13), despite WT levelsof MYO7A (FIG. 11J and FIG. 11L), suggesting that some of the MYO7A maynot be fully functional.

AAV2 MYO7A Dual-Vector Preparations.

The preceding results demonstrate that a single AAV vector is capable ofdelivering functional MYO7A to the RPE and photoreceptor cells in vivo.Because the size of smCBA-MYO7A is ˜2 kb larger than the nominalcarrying capacity of an AAV (Grieger and Samulski, 2005), thistransduction may involve undefined fragmentation of the smCBA-MYO7A cDNAfollowed by reassembly of plus and minus cDNA strands after delivery tothe cell as shown for other large genes (Dong et al., 2010; Lai et al.,2010; Wu et al., 2010). To evaluate whether two AAV vectors containingdefined, overlapping fragments of MYO7A cDNA (1365 bases) were alsocapable of mediating full-length MYO7A expression, an AAV2-baseddual-vector system (FIG. 14A-1 and FIG. 14A-2) was developed. Twoseparate lots of the AAV2-MYO7A(dual vector) were prepared, eachcontaining equal concentrations of AAV2-smCBA-MYO7A(5′-half) andAAV2-MYO7A(3′-half). The titer of the first lot contained 2.5×10¹²particles/mL of each vector, and the second lot contained 4×10¹²particles/mL.

MYO7A Expression with AAV2 Dual Vectors.

Western blot analysis of primary cultures of Myo7a-null RPE cells,infected with AAV2-MYO7A(dual vector) of either lot, showed that thecells expressed a MYO7A-immunolabeled polypeptide of comparable mass tothat of WT MYO7A (FIG. 14B). However, the expression level of MYO7A inthe Myo7a-null RPE cells was significantly less than that found inprimary cultures of Myo7a^(+/−) RPE cells (cf. lanes 2 and 3 in FIG.14B), unlike that found for the single AAV2 or AAV5 vectors (FIG. 10B).Quantitative analysis of western blots showed that Myo7a-null RPE cells,transduced with the single vectors (1×), AAV2-MYO7A or AAV5-MYO7A, orwith AAV2-MYO7A(dual vector), expressed MYO7A at levels that were 82%,111%, and 10%, respectively, of the level of MYO7A in Myo7a^(+/−) RPEcells.

FIG. 16 is a Western blot using the same dual-vector system as aboveexcept in an AAV8 serotype. FIG. 16 shows expression level of myo7Ausing the dual-vector system that was nearly equivalent to the wild typemyo7A expression level. While the reasons for the discrepancy areunclear, much better results were obtained by the inventors using thedual-vector systems than were obtained by several outside collaborators.Wild type like levels of MYO7A expression was observed in shaker-1retinas following injection with dual-AAV8(Y733F) vectors. Thus, verygood expression of MYO7A with the dual AAV platform has been achieved.

Immunofluorescence of primary Myo7a-null RPE cells, infected withAAV2-MYO7A(dual vector), showed that a few cells scattered throughoutthe culture exhibited very high levels of MYO7A, but all other cellscontained insignificant levels (FIG. 14C, FIG. 14D, and FIG. 14E). Thecells overexpressing MYO7A typically had altered morphology, suggestingthat the high levels of MYO7A may be toxic. Similarly,immunofluorescence of ARPE19 cells, infected with AAV2-MYO7A(dualvector), resulted in a minority of cells that were labeled intenselywith MYO7A antibody, with most of the cells appearing to express onlyendogenous levels of MYO7A (FIG. 14F and FIG. 17F).

Immunolabeling of retinas, prepared 3 weeks after subretinal injectionwith AAV2-MYO7A(dual vector) of either lot, also showed only a few RPEcells and photoreceptor cells with clear MYO7A expression, althoughsignificant overexpression was not evident in this in vivo experimentImmunogold particle counts from images of ultrathin sections were usedto quantify the level of MYO7A expression in Myo7a-null retinas thatwere treated with the second lot of AAV2-MYO7A(dual vector). Within 1.4mm of the injection site, MYO7A immunolabeling of the connecting ciliumand pericilium of the photoreceptor cells was a mean of 48% of that inWT retinas: 2.8 particles/μm (n=3 retinas) compared with 6.5particles/μm for WT (n=3 retinas). The mean label density inapical-basal sections of the RPE was 35% of that in WT retinas: 11particles/100 μm² compared with 31 particles/100 μm² for WT. However, itwas clear that these lower means were achieved by some cells expressingnear normal amounts of MYO7A and the majority expressing very little;over half the cells had fewer than 10 particles/100 μm² (FIG. 14G).

Correction of Myo7a-Mutant Phenotypes with AAV2 Dual Vectors.

Eyes were analyzed for correction of melanosome localization and ciliaryopsin distribution within 1.4 mm of the injection site. With either lotof AAV2-MYO7A(dual vector), some RPE cells (29% for lot 1 treatment [n=6retinas], 35% for lot 2 treatment [n=9 retinas]) were observed to have anormal apical melanosome distribution, but most of the cells in thisregion retained the Myo7a-mutant phenotype, resulting in a mosaic effect(FIG. 15A) that contained a much lower proportion of corrected cellsthan that observed with a 1:100 dilution of either of the singlevectors. The only correction observed in 3 eyes injected with a 1:10dilution of AAV2-MYO7A(dual vector) (first lot), was in 18% of the RPEcells in one of the retinas. With full-strength of AAV2-MYO7A(dualvector) (second lot), opsin immunogold density averaged 3.2±0.4particles/μm of cilium length, which was reduced from untreated retinas(4.2±0.8 particles/μm; p=0.003), but still greater than WT levels(1.1±0.2 particles/μm), suggesting that most cells were not corrected.

Using immunoelectron microscopy, a correlation between phenotypecorrection and the expression level of MYO7A was identified (determinedby the mean concentration of immunogold particles in an apical-basalsection of each RPE cell) (FIG. 15B, FIG. 15C, FIG. 15D, and FIG. 15E).From the eyes injected with AAV2-MYO7A(dual vector) (second lot), it wasshown that the corrected RPE cells contained a mean of 108% of the WTlevel of MYO7A (the minimum level was 82%). RPE cells that were notcorrected contained a mean of 26% of the WT level of MYO7A (the maximumlevel was 92%). While these data showed that higher expression of MYO7Ais correlated with phenotype correction (FIG. 15F), it also indicatedthat some of the labeled MYO7A protein was not functional, given thatmelanosomes are localized normally in mice that are heterozygous for theMyo7a-null allele and have only ˜50% of the WT level of MYO7A.

Expression of MYO7A with Simple Overlap Vectors.

AAV2-based simple overlap vectors were evaluated in vitro at a varietyof MOIs to evaluate how the concentration of vector pairs related toMYO7A expression. How levels of MYO7A changed over time was alsoevaluated in infected cells. HEK293 cells were infected with simpleoverlap vector pairs packaged in AAV2(tripleY-F) vector (FIG. 23A). Apreliminary co-infection with AAV2(tripleY-F) simple overlap vectors(MOI of 10,000 for each vector) indicated that MYO7A is expressed, andthat migration of the protein on gel is identical to a full-lengthtransfection control (FIG. 23A). Coinfection at MOIs of 400, 2000, and10,000 of each vector shows that the efficiency of the simple overlapsystem is proportional to the amount of 5′ and 3′ vectors used (FIG.23B). MYO7A expression increased as a function of incubation time up to5 days postinjection in HEK293 cells (FIG. 23C). The visible expressiondecline was because of a reduction of viable cells in the culture vesselat the later times.

Comparison of fAAV-MYO7A to Dual-AAV-MYO7A Expression and Evaluation ofAAV Serotype Efficiency.

Previously, it was shown that fAAV-MYO7A was able to ameliorate theretinal phenotype of the shaker1 mouse (Colella et al., 2013; Lopes etal., 2013; Trapani et al., 2013). To provide a basis for comparisondual-AAV-vector expression was evaluated relative to fAAV in vitro.After infection in HEK293 cells, all dual-vector systems expressed MYO7Amore efficiently than fAAV (FIG. 24). The AP hybrid platform showed thestrongest expression, followed by the simple overlap system.

Other studies have shown, in the context of a conventionally sized DNApayload, that the transduction efficiency and kinetics ofAAV2(tripleY-F) vectors are increased relative to standard AAV2 both invitro and in vivo (Li et al., 2010; Markusic et al., 2010; Ryals et al.,2011). The efficiency of AAV2 versus AAV2(tripleY-F) dual vectors wasdirectly compared in HEK293 cells. Surprisingly, standard AAV2-mediatedMYO7A expression was higher than that seen with titer-matchedAAV2(tripleY-F) (FIG. 24). Identical results were obtained whencomparing different AAV2 and AAV2(tripleY-F) dual-vector preparationpackaged with identical vector plasmid.

Comparison of Relative Efficiencies and Specificity of Full-Length MYO7AExpression.

To quantitatively evaluate the relative expression efficiencies of thedual-vector platforms and to assess specificity of full-length protein,HEK293 cells were infected with either the 5′ and 3′ AAV2-based vectorpairs combined or the corresponding 5′ vector alone. An additionalhybrid vector pair was included that incorporated native MYO7A intronicsequence (intron 23) that served as overlapping sequence and providedappropriate splicing signals. All 5′ vectors produced low amounts of adefined, less than full-length peptide detectable on Western blot withthe exception of the simple overlap vector (FIG. 25A). However, thetrans-splicing and the AP hybrid platforms revealed a distinct decreaseof this undesired product when the 3′ vector was added to the sample(FIG. 25A). The native intron hybrid platform also showed this secondaryband on Western blots, again suggestive of a truncated proteinoriginating from the 5′ vector alone. In contrast to all other platformstested, this band intensity increased with the addition of the 3′vector. Each platform's relative ability to promote reconstitution wascompared by quantifying the amount of 5′ vector-mediated truncatedprotein product in the presence or absence of the respective 3′ vector(FIG. 25B). Full-length MYO7A expression on Western blot was thenquantified relative to transfection control (FIG. 25C). APhybrid-mediated MYO7A was the strongest followed by simple overlap,trans-splicing, and native intron hybrid (FIG. 25C).

Characterization of the Overlap/Splice Region of the Expressed MYO7A.

To characterize the fidelity of the mRNA arising from dual vectors,HEK293 cells were infected with dual vectors and RNA extracted, reversetranscribed, and subjected to PCR utilizing primers binding upstream ofthe overlap region and in the bGH polyA signal region producing a 4.5 kbPCR fragment (FIG. 26A). An identically treated sample not containingreverse transcriptase was used as control for chromosomal DNAcontamination. Plasmid containing the full-length MYO7A coding sequencewas used as positive control for PCR. A preliminary screen ofAAV-mediated MYO7A mRNA was performed by analyzing the pattern offragment migration on agarose gel following restriction endonucleasedigests with PpuMI and BglII (FIG. 26A). Identical banding patterns,consistent with the predicted pattern (PpuMI: 1591, 876, 556, 548, 541,238, 168, 42, and 36 bp; BglII 1335, 1074, 827, 583, 360, 272, and 146bp), were observed following digests of amplicons from each dual-vectorplatform tested, indicating that no gross alterations(deletions/insertions) occurred as a consequence of either homologousrecombination of vector pairs and/or RNA splicing (FIG. 26B). To furthercharacterize the fidelity of the overlap region, a fragment containingthe complete overlap area (1829 bp) was restricted and cloned into pUC57(FIG. 26A). Sequencing results of 10 clones picked at random per vectorplatform revealed that the overlap region was 100% identical to theconsensus/predicted MYO7A sequence (FIG. 26C). This indicated that, inthe context of the simple overlap platform, homologous recombination wasaccurate. Additionally, in the context of trans-splicing vectors,accurate splicing occurred. Finally, for the AP hybrid vectors, acombination of accurate homologous recombination and/or splicing tookplace. To determine whether this protocol was capable of detectingaberrant sequence in reconstituted MYO7A, a sequence that containedeither an insertion of a HindIII recognition site (TAGC) at position2635 or a point mutation (T-C) at position 2381 was also generated.

MYO7A Expression Mediated by Dual Vectors in Mouse Retina.

To investigate the expression of MYO7A from the two best performingdual-vector platforms in vivo, C57BL/6J mice were subretinally injectedwith 1×10¹⁰ vector genomes per eye of simple overlap and AP hybridsystems packaged in AAV8(733) and analyzed 4 weeks later by Western blotand immunohistochemistry. AAV8(733)-fAAV-MYO7A vector was also injectedto provide a basis for comparison. To distinguish between endogenousMYO7A and exogenous expression mediated by vectors, sequence coding foran HA tag was added to the C′ terminus of the MYO7A cDNA in allconstructs. Resulting retinas were immunostained for HA to reveal thatfAAV vector along with both dual-vector platforms mediated expression ofMYO7A in photoreceptors and RPE. A recent report concluded that simpleoverlap vectors were more efficient for gene transfer to the RPE thanphotoreceptors (Trapani et al., 2013). Simple overlap-mediated MYO7Aexpression was observed in both RPE and photoreceptors. In contrast toprevious results showing “spotty” MYO7A expression mediated byAAV2-based simple overlap vectors (Lopes et al., 2013), it was found,when packaged in AAV8(733), that simple overlap vectors mediated MYO7Aexpression in the majority of RPE and photoreceptor cells. Photoreceptordegeneration/outer nuclear layer thinning was apparent in eyes injectedwith the AP hybrid vector system. Despite the observed degeneration, APhybrid-mediated MYO7A was clearly detected in residual PR cell bodiesand RPE and was sufficient to be detected by immunoblot. By Western blotanalysis using HA antibody, simple overlap-mediated MYO7A was present injust detectable amounts. In contrast, fAAV-mediated protein levels wereinsufficient to be detected in this assay. Using an antibody againstMYO7A, immunoblot of WT mouse retina revealed that both endogenous MYO7Aand dual-vector-mediated, HA-tagged MYO7A migrated similarly.

DISCUSSION

In this example, it was shown that dual AAV vectors with defined geneticpayloads can be used to deliver a large transgene in vitro and in vivo.The initial experiments using the simplest of all dual-vector platformsrevealed that efficiency of AAV2-based simple overlap vectors isproportional to the amount of 5′ and 3′ vectors used and that MYO7Aexpression mediated by this system increased as a function of incubationtime in HEK293 cells. Next, three distinct dual-vector platforms wereevaluated and compared to single, fragmented fAAV vector in vitro. Alldual vectors analyzed drove higher levels of MYO7A expression than fAAV.Of all platforms tested, a hybrid vector system containing overlapping,recombinogenic sequence and splice donor/acceptor sites from the AP gene(AP hybrid) was the most efficient.

Regarding the specificity with which the dual-vector platforms expressthe correct-sized gene product, it was noted in vitro thattrans-splicing and hybrid dual-vector platforms generated an additionalband of lower molecular weight as detected by immunoblot (monoclonalantibody used was raised against the amino terminus MYO7A). Theexpression of this truncated product was much more pronounced forinfections with 5′ vectors alone. What might account for this additionalband? After entry into the host cell, the virus capsid is removed andthe single-stranded DNA payload is released. The ITRs carried by thesingle strand serve as primer for DNA polymerases to produce a doublestrand. The resulting circular intermediates consist mainly of monomersthat, over time, convert into multimeric concatemers throughintermolecular recombination (Duan et al., 1998; Yang et al., 1999). Thedual-vector systems created in this study utilize this strategy toachieve full-length protein expression. A limiting factor lies in thefact that the highly recombinogenic ITRs flanking the expressioncassettes are identical in nature leading to a random recombination andconsequently a random orientation of the vector parts relative to eachother. This random recombination inevitably results in reducedefficiency because only concatemers that have the two vector parts in5′-3′ orientation are able to express the full-length protein. Thisconcatemerization over time is consistent with the observation that theamount of single-vector product is reduced in favor of the full-lengthprotein when both 5′ and 3′ vectors are combined. Interestingly, thesimple overlap system does not generate truncated product, even whenonly the 5′ vector is used for infections. In contrast to thetrans-splicing and hybrid vectors, there is virtually no interveningsequence between the end of the MYO7A coding sequence and the right-handITR. It may be that splice donor sequences enhance the likelihood oftruncated product through some as-yet-to-be-determined mechanism.

A number of strategies have been devised to overcome the issue of randomconcatemerization and thereby increase specificity as well as efficiencyof these dual-vector platforms. First, the addition of ahighly-recombinogenic sequence such as that used in the AP hybrid vectorhere has resulted in significantly increased protein expression comparedwith the trans-splicing system. Ghosh et al. (2011) provide a detailedanalysis of the 270-bp AP sequence used in this study as well as othersequences derived from AP that direct recombination and lead tosignificant improvement over trans-splicing vectors. The finding that APhybrid vectors are more efficient than trans-splicing vectors supportsthat the AP sequence directs at least some of the concatemerizationevents toward the proper orientation with recombination then occurringvia this sequence or via the ITRs. Regardless, with more concatemersproperly aligned, the AP hybrid system mediates a more-efficientexpression of MYO7A. Another approach for directing concatemerization isthe use of single-strand oligonucleotides that are capable of tetheringthe back end of the 5′ vector and the front end of the 3′ vectortogether (Hirsch et al., 2009). However, this strategy requiresefficient delivery of the oligonucleotide to the nucleus of the targetcells timed with the dual vectors. Finally, dual vectors utilizingmismatched ITRs can be used to direct concatemerization in ahead-to-tail orientation (Yan et al., 2005), although the process mayrequire further optimization of the AAV packaging machinery.

Notably, in this study, it was found that the sequence in the overlapregion of all dual vectors tested in vitro was 100% identical to theconsensus/predicted MYO7A sequence. This indicates that homologousrecombination and/or splicing was accurate in each dual-vector platform.

Similar to the in vitro results, the highest levels of MYO7A expressionwas found in retinas of mice subretinally injected with AAV8-based APhybrid vectors (as assessed by probing for HA on Western blot). Notably,no truncated proteins were evident in retinas expressing either simpleoverlap or AP-hybrid mediated MYO7A. The reason for this observeddifference remains to be elucidated but may involve differences in theDNA repair machinery that mediate recombination in actively dividingcells versus post-mitotic photoreceptors/RPE (Hirsch et al., 2013).Dual-vector-mediated MYO7A-HA expression was observed in thephotoreceptors and RPE of WT mice, locations where MYO7A is thought tohave a functional role (Williams and Lopes, 2011). In eyes injected withAP hybrid vectors, marked thinning of the outer nuclear layer wasobserved. It has previously been shown that vector-mediatedoverexpression of MYO7A leads to retinal toxicity (Hashimoto et al.2007). Taken together with the high efficiency of transduction observedin vitro for the AP hybrid platform, the most likely explanation for theobserved pathology is excessive production of MYO7A. Despite the markeddegeneration, significant amounts of AP hybrid-mediated, full-lengthMYO7A-HA were detected on Western blot. As high concentrations ofvectors were used in these experiments, a simple solution to circumventtoxicity could be to reduce vector genomes injected or replace thestrong, ubiquitous smCBA promoter with an endogenous or homologouspromoter, and/or a promoter with attenuated strength. An alternativeexplanation for toxicity of the strong AP hybrid platform is expressionof undesired products, like the observed protein expressed from the 5′vectors alone in vitro. However, it was noted that only full-sizeMYO7A-HA was apparent on Western blot of the AP hybrid-treated retina.

With the goal of developing an AAV-based treatment for USH1B, animalmodels of this disease have provided an abundance of useful information.Similar to previous observations that fAAV-MYO7A and simple-overlap,dual-vectors were capable of restoring melanosome migration and opsinlocalization in the shaker1 mouse (Lopes et al., 2013), a recent studyby an independent lab confirmed the usefulness of the vectors disclosedherein, when it was reported that they were capable of restoring theultrastructural retinal phenotypes in the animal model. Notably, shaker1mice lack retinal degeneration, and the severe functional abnormalitiesseen in USH1B patients (Liu et al., 1997). This fact renders in vivoanalysis of therapeutic outcomes in the shaker1 retina problematic.Alternative animal models for evaluating a treatment for thisdevastating disease may be useful in adaptation of the present methodsto human clinical use.

These results presented here also demonstrated that MYO7A can beefficiently expressed using dual-AAV-vector systems. The platformscontaining overlapping elements, namely, the simple overlap system, andthe AP hybrid system were both highly efficient. AP hybrid vectorsshowed the strongest expression of all systems tested, with littleobservable truncated protein in vitro and none observed in vivo. Simpleoverlap vectors showed good expression and were the most specific (notruncated protein products were observed) even when the 5′-only vectorwas used to infect cells. AAV has emerged as the preferred clinicalvector and it efficiently transduces both photoreceptors and RPE.Because it has now been demonstrated that MYO7A sequence fidelity ispreserved following recombination and/or splicing of dual-AAV-vectorplatforms and because only full-length MYO7A was detectable in mouseretinas injected with dual vectors, the dual-AAV-vector strategypresented herein represents a valid option for the treatment of retinaldisorders associated with mutations in large genes such as USH1B.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

All references cited herein (including publications, patent applicationsand patents) are incorporated by reference to the same extent as if eachreference was individually and specifically incorporated by reference,and was set forth in its entirety herein.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable order,unless otherwise indicated herein, or unless otherwise clearlycontradicted by context.

The use of any examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illustrate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of ordinary skill in the art that variations may be applied to thecompositions and/or methods disclosed herein, and/or to the steps or thesequence of steps of the methods described herein without departing fromthe concept, spirit and/or scope of the invention. More specifically, itwill be apparent that certain agents that are chemically- and/orphysiologically-related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A polynucleotide vector system comprising i) afirst AAV vector polynucleotide comprising an inverted terminal repeatat each end of the polynucleotide, and between the inverted terminalrepeats a promoter followed by a partial coding sequence that encodes anN-terminal part of a selected full-length polypeptide, and ii) a secondAAV vector polynucleotide comprising an inverted terminal repeat at eachend of the polynucleotide, and between the inverted terminal repeats apartial coding sequence that encodes a C-terminal part of the selectedfull-length polypeptide, and optionally followed by a polyadenylation(pA) signal sequence, wherein the polynucleotide sequence encoding thepolypeptide sequence in the first and second AAV vectors comprisespolynucleotide sequence that overlaps.
 2. A polynucleotide vector systemcomprising i) a first AAV vector polynucleotide comprising an invertedterminal repeat at each end of the polynucleotide, and between theinverted terminal repeats a promoter followed by a partial codingsequence that encodes an N-terminal part of a selected full-lengthpolypeptide followed by a splice donor site and an intron, and ii) asecond AAV vector polynucleotide comprising an inverted terminal repeatat each end of the polynucleotide, and between the inverted terminalrepeats an intron and a splice acceptor site for the intron, andoptionally followed by a partial coding sequence that encodes aC-terminal part of the selected full-length polypeptide, followed by apolyadenylation (pA) signal sequence, wherein the intron sequence in thefirst and second AAV vectors comprises polynucleotide sequence thatoverlaps.
 3. The polynucleotide vector system of claim 1, wherein thepolypeptide-encoding sequence overlap of the first and second AAVvectors is between about 500 and about 3000 nucleotides.
 4. Thepolynucleotide vector system of claim 2, wherein the intron sequenceoverlap of the first and second AAV vectors is about 50 to about 500nucleotides.
 5. The polynucleotide vector system of claim 2, wherein thepromoter is selected from the group consisting of a chimeric CMV β actin(smcBA) promoter, a human myosin 7a gene-derived promoter, a conetransducin α (TαC) gene-derived promoter, a rhodopsin promoter, acGMP-phosphodiesterase β-subunit promoter, a human rhodopsin kinase(hGRK1) promoter, a rod specific IRBP promoter, a RPE-specificvitelliform macular dystrophy-2 [VMD2] promoter, and combinationsthereof.
 6. The polynucleotide vector system of claim 2, wherein theselected full-length polypeptide is encoded by a gene of about 5 Kb toabout 10 Kb in length.
 7. The polynucleotide vector system of claim 1,wherein the polypeptide encoded is human myosin VIIa, or a functionalfragment thereof.
 8. The polynucleotide vector system of claim 5,wherein the human myosin VIIa polypeptide comprises the amino acidsequence of SEQ ID NO:6 or SEQ ID NO:8, or a functional fragmentthereof.
 9. The polynucleotide vector system of claim 1, wherein thefirst AAV vector polynucleotide comprises the nucleotide sequence of SEQID NO:1, or a functional fragment or variant thereof, and the second AAVvector polynucleotide comprises the nucleotide sequence of SEQ ID NO:2,or a functional fragment or variant thereof.
 10. The polynucleotidevector system of claim 2, wherein the first AAV vector polynucleotidecomprises the nucleotide sequence of SEQ ID NO:3, or a functionalfragment or variant thereof, and the second AAV vector polynucleotidecomprises the nucleotide sequence of SEQ ID NO:4, or a functionalfragment or variant thereof.
 11. A virus or an infectious viral particlecomprising the first AAV vector polynucleotide or the second AAV vectorpolynucleotide of claim
 1. 12. The virus or virion of claim 11,characterized as an adeno-associated virus (AAV) or an infectious AAVviral particle.
 13. The virus or virion of claim 11, comprising one ormore tyrosine-to-phenylalanine (Y→F) mutations in a capsid protein ofthe virus or virion.
 14. The virus or virion of claim 12, comprising oneor more tyrosine-to-phenylalanine (Y→F) mutations in a capsid protein ofthe virus or virion at amino acid position 730, or 733, respectively inthe AAV2 or AAV8 capsid.
 15. An isolated host cell comprising thepolynucleotide vector system of claim
 1. 16. A method for treating orameliorating a disease or condition in a human or animal, comprisingproviding in one or more cells of the human or animal, thepolynucleotide vector system of claim 1, wherein the full-lengthpolypeptide provides for treatment or amelioration of the disease orcondition and is expressed in the one or more cells.
 17. The method ofclaim 9, wherein the disease or condition is Usher syndrome.
 18. Themethod of claim 9, wherein the polypeptide is human myosin VIIa, or afunctional fragment thereof.
 19. The method of claim 11, wherein thehuman myosin VIIa polypeptide comprises the amino acid sequence of SEQID NO:6 or SEQ ID NO:8, or a functional fragment thereof.
 20. A methodfor expressing a selected polypeptide in a cell, comprisingincorporating in the cell, the polynucleotide vector system of claim 1,and expressing the sequences of the vector system in the cell.
 21. Themethod of claim 19, wherein the cell is a photoreceptor cell, a conecell, a rod cell, a retinal cell, or any combination thereof.
 23. Themethod of claim 19, wherein the cell is located in vivo.
 24. The methodof claim 19, wherein the cell is located ex vivo.
 25. The method ofclaim 19, wherein the polypeptide is a therapeutic polypeptide or afunctional polypeptide.